prompt
stringlengths 841
87k
| target
stringlengths 220
5.76k
| input_tokens
int64 256
16.4k
| target_tokens
int64 96
1.02k
| subset
stringclasses 17
values | language
stringclasses 5
values |
|---|---|---|---|---|---|
Summarize: PRIOR APPLICATION [0001] This application claims the benefit of U.S. Provisional Application Ser. No. 60/612,162, filed Sep. 23, 2004 entitled “MACHINE FOR LAYING SOD”. FIELD OF THE INVENTION [0002] This invention relates to a sod laying machine. The machine of the invention can also be used for laying other landscaping products, and other types of products. BACKGROUND OF THE INVENTION [0003] It has been common practice for approximately the last half century to cut sod from a field of sod, stack the cut sod on a pallet (usually in the form of rolls or slabs), and then to transport the pallet (usually by truck) to a site where the sod is to be laid. The steps involved in the foregoing procedure have been largely automated, and improved efficiencies in cutting, stacking and handling the sod are continually being developed. [0004] However the task of laying the sod on ground to be sodded remains almost entirely an intensive manual labor task. Various machines have been developed for laying sod, in an attempt to automate the process, but so far as is known, no one has been able to develop a successful machine for laying small rolls and slabs of sod. All of the sod laying machines introduced to date have been too complicated, or too expensive, or have suffered from other major limitations which made them unacceptable in the marketplace. For example, in many instances the machines were too large to travel through the narrow passageways which are often the only way to access yards and other locations where sod is to be laid. SUMMARY OF THE INVENTION [0005] It is therefore an object of present invention, in one of its aspects, to provide a machine which is relatively simple and inexpensive, and which facilitates the laying of sod and reduces the time spent in manually laying the sod. In one aspect the invention provides a vehicle for facilitating the laying of a landscaping material on the ground, said vehicle comprising: (a) first and second movable supports for supporting said vehicle on the ground and for driving said vehicle over the ground, said movable supports being spaced apart laterally to leave an open-ended space there between, (b) a forklift located in said space and having fork tines to support a pallet laden with said landscaping material, and (c) a pair of platforms, one located over each of said movable supports and each adapted to support a person such that said person while on said platform can reach landscaping material on said pallet and can place said landscaping material on the ground. [0006] Further objects and advantages of the invention will appear from the following description, taken together with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0007] In the drawings: [0008] FIG. 1 is a perspective view of a machine according to the invention; [0009] FIG. 2 is another perspective view of the machine of FIG. 1, with a pallet in place thereon, the pallet being close to the ground; [0010] FIG. 3 is a perspective view similar to FIG. 2 but with the pallet raised; [0011] FIG. 4 is an end view of the machine of FIG. 1 with the pallet in position thereon; [0012] FIG. 5 is a side view of the machine of FIG. 1 ; [0013] FIG. 6 is a perspective view from above of the controls for the machine of FIG. 1 ; [0014] FIG. 7 is an end view of the machine of FIG. 1 ; [0015] FIG. 8 is a diagrammatic side view of a portion of a preferred forklift mechanism for the FIG. 1 machine; and [0016] FIG. 9 is a view similar to that of FIG. 8 but showing the forklift mechanism in position to pick up a pallet. DETAILED DESCRIPTION OF THE INVENTION [0017] As shown in the drawings, the machine 10 according to the invention, is preferably a tracked vehicle, having a pair of relatively wide tracks 12. The tracks 12 are of conventional design, typically being formed of a rubberized material with short projecting ridges 14 or knobs for traction, and are supported on a conventional support structure 16 at each side of the machine 10. The track support structure 16 includes the usual guide wheels 18 and sprockets 20 to guide and drive the tracks 12, and a track support frame 21. The tracks 12 are relatively wide, so as to apply a low pressure to the ground over which they travel, and are spaced laterally apart, leaving an open-ended space 22 between them. The support structures 16 for each track 12 are joined by a cross-frame structure 24 at one end 26 of the machine 10. Any suitable track design can be used. [0018] Located between the tracks 12, in the open-ended space 22, is a conventional forklift 30. Forklift 30 typically has a fork 32 with a pair of tines 34, which can be raised and lowered on a mast 36, using any desired conventional elevating mechanism. For example, since only a fairly small lift is required for the tines 34, a simple piston and cylinder mechanism 38 can be used to raise and lower the fork. [0019] The fork 32 can be used to engage and support a pallet 40 on which sod (not shown) is stacked. The sod can be stacked on pallet 40 in the form of folded or unfolded slabs, or in rolls, as desired. [0020] Located on each side of the open-ended space 22, over each track 12, is a platform 42. The two platforms 42, one on each side of the machine 10, are mirror images of each other. Each platform includes a horizontal perforated metal foot plate 44 which extends lengthwise of the machine for approximately the length of the pallet, and also extends laterally outwardly slightly beyond each track 12, as best shown at 45 in FIG. 7. Each platform 42 also includes a vertical upwardly extending guard plate 46 at the inner edge of the foot plate 44, to prevent a person standing on the foot plate from inadvertently stepping through the narrow space between the inner edge of the foot plate 44 and the sod laden pallet 40. Each platform 42 is supported adjacent the open end of the space 22 by a laterally extending support beam 48 attached to a vertical downwardly extending flange 50. The flange 50 is connected, e.g. by a screw 52, to the track support frame 21. At the other end of the machine 10, the platforms 42 are connected to a pair of railing structures 54, one on each side of the machine 10. The railing structures 54 are connected to two laterally spaced posts 56 which form vertical guides for the fork 32. The posts 56 are connected to the cross-frame structure 24 by a set of beams 58 ( FIG. 5 ). [0021] The driver of the machine 10 stands on a platform 60 which extends from the end of the machine opposite from the open-ended space 22. The platform 60 may be connected very simply to the machine 10, e.g. by a pair of support bars 62 connected between the cross-frame support structure 24 and the outer end of the platform 60. [0022] The machine 10 includes a motor and hydraulic pump (hidden from view by covers) to move the machine and operate the forklift 30. Conventional controls 66 are provided to operate the machine 10, by driving each track 12 forwardly or rearwardly at desired speed to propel the machine and to steer it, and also to control the forklift 30. Since the machine 10 normally operates at low speed, with numerous stops and starts, it preferably has a conventional hydrostatic or other automatic transmission. [0023] In use, the fork 32 will support a pallet 40, the pallet being loaded with sod. Because the machine 10 is small, it can be driven into yards and other restricted spaces which cannot be accessed by larger machines. Even on wet ground, the wide tracks will cause minimal damage. [0024] A sod laying person will stand on each platform 42 at each side of the machine 10, and as the machine 10 travels over the ground on which sod is to be laid, the sod laying persons will be able to reach sod pieces stacked on the pallet 40 and will be able to throw the sod pieces onto the ground on each side of the machine 10, or in front of the machine (the front being the pallet). After the sod laying persons have thrown an appropriate number of sod pieces onto the ground from each side of the machine 10, these persons can easily step off the machine 10 (since the platforms 42 are low) and can use rakes or other long handled tools to place the pieces of sod in the exact locations desired. In many cases this task can also be performed by other people already on the ground. However, if the persons on the platforms 42 have stepped off them to adjust the location of the sod pieces on the ground, then they can easily step back on the platforms 42 to lay further pieces of sod as the machine 10 moves through the area where sod is being laid. [0025] When a pallet 40 full of sod is initially placed on the machine 10, and when sod is being laid, the pallet 40 will normally be in a low position, close to the ground, so that the top pieces of sod stacked on the pallet 40 are readily accessible to the sod laying persons standing on the platforms 42 on each side of the pallet 40. As the sod at the top of the pallet 40 is laid, the fork 32 and hence the pallet 40 will be raised, to bring the new and lower top of the sod stacked on the pallet within easy reach of the persons standing on the platforms 42. [0026] Because the machine 10 provides a platform 42 on each side of the pallet 40, with ready access to the sod stacked on the pallet, and because the platforms 42 are low (close to the ground) and access from them to the ground in a forward or sideways direction is not obstructed, the machine 10 makes the task of lifting sod pieces off the pallet 40 and placing them in the desired locations on the ground very simple and enables this task to be performed rapidly and efficiently by persons standing on the platforms 42. In addition, because the machine 10 is small and lightweight, it can easily pass through small passageways and any damage which it may do to earth which has been prepared to receive sod will usually be minimal. [0027] Because the platforms 42 are over the tracks 12, and are next to the sod to be laid (stored on pallet 40 ), and because the platforms 42 extend laterally outwardly beyond the tracks 12, access to the ground by the persons standing on the platforms 42 is maximized, and interference with this access by the tracks 12 is minimized. [0028] It will be understood that various changes can be made in the machine described. For example, while a tracked vehicle is preferred because of its minimal impact on soft earth, a wheeled vehicle could also be used, provided that the tires are relatively wide and low and do not concentrate pressure on the ground. In addition, while the machine 10 has been described for use in laying sod, it can be used to lay other landscaping products, e.g. other plant materials, or even paving stones, or other non-landscaping products. [0029] While a conventional forklift mechanism 30 can be used, a forklift mechanism such as that shown in FIGS. 8 and 9 is preferred. In the forklift mechanism there shown, the fork 32 is carried on a fork carriage 100 having four guide wheels 102 (only two are shown) which ride in the channels formed by vertical guideposts 56. These channels, indicated at 104 in FIGS. 8 and 9, have a downwardly and rearwardly extending bottom portion 106. Thus, as the forks are lowered, the lower fork carriage wheel 102 will move into the downwardly and rearwardly extending channel portion 106 and will move to the rear, as shown in FIG. 9. This rearward movement of lower wheel 102 tilts the fork tines 34 downwardly and forwardly, thus facilitating pick up of a pallet laden with sod. Since no tilt cylinder or associated mechanism is required for the downward tilt described, the cost of the tilt mechanism is kept very low. [0030] It will be appreciated that various changes can be made in the machine as described, and all such changes are intended to be included in the invention. | Summary: A machine to facilitate sod laying, having a pair of tracks spaced apart to define an open-ended space between them, with a low platform over each track and a forklift supporting a pallet load of sod or other materials located in the space between the tracks. Persons laying sod can stand on the platforms and can without obstruction place sod or other materials from the pallet on the forklift onto the ground. The forklift carriage has wheels which are guided by a pair of channels having downwardly and rearwardly extending lower portions, so that when the wheels enter the lower portions, the fork tines will be tilted downwardly and forwardly without needing a costly tilt cylinder. | 3,119 | 155 | big_patent | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Native American Indian Education Act''. SEC. 2. FINDINGS AND PURPOSE. (a) Findings.--Congress finds the following: (1) Nontribal colleges that serve Native American Indian students have a valuable supplemental role to that provided by tribally controlled community colleges in making available educational opportunities to Native American Indian students. (2) Some 4-year colleges serve Native American Indian students by providing tuition-free education, with the support of the State in which the institutions are located, as mandated by Federal statute, to hundreds of Native American Indian students in fulfillment of a condition under which the United States provided land and facilities for colleges to a State or college. (3) The value of the Native American Indian student tuition waiver benefits contributed by these colleges and the States that support them today far exceeds the value of the original grant of land and facilities. (4) The ongoing financial burden of meeting this Federal mandate to provide tuition-free education to Native American Indian students is no longer equitably shared among the States and colleges because it does not distinguish between Native American Indian students who are residents of the State or of another State. (5) In fiscal year 2012, the State of Colorado paid approximately $13,000,000 in tuition fees to support the education of Native American Indian students at Fort Lewis College in Colorado. In the State of Minnesota, the University of Minnesota waived $2,600,000 in tuition for Native American Indian students in fiscal year 2012. (6) Native American Indian student tuition waiver benefits are now at risk of being terminated by severe budget constraints being experienced by these colleges and the States which support them. (b) Purpose.--It is the purpose of this Act to ensure that Federal funding is provided in order to relieve constrained State education budgets and to support and sustain the longstanding Federal mandate requiring colleges and States to waive, in certain circumstances, tuition charges for Native American Indian students admitted to an undergraduate college program, including the waiver of tuition charges for Native American Indian students who are not residents of the State in which the college is located. SEC. 3. STATE RELIEF FROM FEDERAL MANDATE. Part A of title III of the Higher Education Act of 1965 (20 U.S.C. 1057 et seq.) is amended by inserting after section 319 the following: ``SEC. 319A. STATE RELIEF FROM FEDERAL HIGHER EDUCATION MANDATE. ``(a) Amount of Payment.-- ``(1) In general.--Subject to paragraphs (2) and (3), for fiscal year 2014 and each succeeding fiscal year, the Secretary shall pay to any eligible college an amount equal to the charges for tuition for such year for all Native American Indian students who-- ``(A) are not residents of the State in which the college is located; and ``(B) are enrolled in the college for the academic year ending before the beginning of such fiscal year. ``(2) Eligible colleges.--For purposes of this section, an eligible college is any institution of higher education serving Native American Indian students that provides tuition-free education as mandated by Federal statute, with the support of the State in which it is located, to Native American Indian students in fulfillment of a condition under which the college or State received its original grant of land and facilities from the United States. ``(3) Limitation.--The amount paid to any eligible college for each fiscal year under paragraph (1) may not exceed the amount equal to the charges for tuition for all Native American Indian students of that college who were not residents of the State in which the college is located and who were enrolled in the college for academic year 2012-2013. ``(b) Treatment of Payment.--Any amounts received by an eligible college under this section shall be treated as a reimbursement from the State in which the college is located, and shall be considered as provided in fulfillment of any Federal mandate upon the State to admit Native American Indian students free of charge of tuition. ``(c) Rule of Construction.--Nothing in this section shall be construed to relieve any State from any mandate the State may have under Federal law to reimburse a college for each academic year-- ``(1) with respect to Native American Indian students enrolled in the college who are not residents of the State in which the college is located, any amount of charges for tuition for such students for such academic year that exceeds the amount received under this section for such academic year; and ``(2) with respect to Native American Indian students enrolled in the college who are residents of the State in which the college is located, an amount equal to the charges for tuition for such students for such academic year. ``(d) Definition of Native American Indian Students.--In this section, the term `Native American Indian students' includes reference to the term `Indian pupils' as that term has been utilized in Federal statutes imposing a mandate upon any college or State to provide tuition-free education to Native American Indian students in fulfillment of a condition under which the college or State received its original grant of land and facilities from the United States.''. SEC. 4. OFFSET. (a) In General.--Notwithstanding any other provision of law, $15,000,000 in appropriated discretionary funds are hereby rescinded, on a pro rata basis, by account, from all available unobligated funds. (b) Implementation.--The Director of the Office of Management and Budget shall determine and identify from which appropriation accounts the rescission under subsection (a) shall apply and the amount of such rescission that shall apply to each such account. Not later than 60 days after the date of enactment of this Act, the Director of the Office of Management and Budget shall submit a report to the Secretary of the Treasury and Congress of the accounts and amounts determined and identified for rescission under the preceding sentence. (c) Exception.--This section shall not apply to the unobligated funds of the Department of Defense, the Department of Veterans Affairs, or the Department of Education, or any unobligated funds available to the Department of the Interior for the postsecondary education of Native American Indian students. | Title: Native American Indian Education Act Summary: Native American Indian Education Act - Amends the Higher Education Act of 1965 to direct the Secretary of Education to pay institutions of higher education the out-of-state tuition of their Indian students if the schools are required to provide a tuition-free education, with the support of their state, to Native American Indian students in fulfillment of a condition under which the college or state received its original grant of land and facilities from the federal government. Limits that payment each fiscal year to the institution's total out-of-state tuition for Native American Indian students in academic year 2012-2013. Treats such payments as reimbursements to such institutions from their states. Rescinds unobligated discretionary appropriations to offset the costs of this program. | 1,433 | 175 | billsum | en |
Summarize: Actus Tertius. Flourish. Enter the Duke of Florence, the two Frenchmen, with a troope of Souldiers. Duke. So that from point to point, now haue you heard The fundamentall reasons of this warre, Whose great decision hath much blood let forth And more thirsts after 1.Lord. Holy seemes the quarrell Vpon your Graces part: blacke and fearefull On the opposer Duke. Therefore we meruaile much our Cosin France Would in so iust a businesse, shut his bosome Against our borrowing prayers French E. Good my Lord, The reasons of our state I cannot yeelde, But like a common and an outward man, That the great figure of a Counsaile frames, By selfe vnable motion, therefore dare not Say what I thinke of it, since I haue found My selfe in my incertaine grounds to faile As often as I guest Duke. Be it his pleasure Fren.G. But I am sure the yonger of our nature, That surfet on their ease, will day by day Come heere for Physicke Duke. Welcome shall they bee: And all the honors that can flye from vs, Shall on them settle: you know your places well, When better fall, for your auailes they fell, To morrow to'th the field. Flourish. Enter Countesse and Clowne. Count. It hath happen'd all, as I would haue had it, saue that he comes not along with her Clo. By my troth I take my young Lord to be a verie melancholly man Count. By what obseruance I pray you Clo. Why he will looke vppon his boote, and sing: mend the Ruffe and sing, aske questions and sing, picke his teeth, and sing: I know a man that had this tricke of melancholy hold a goodly Mannor for a song Lad. Let me see what he writes, and when he meanes to come Clow. I haue no minde to Isbell since I was at Court. Our old Lings, and our Isbels a'th Country, are nothing like your old Ling and your Isbels a'th Court: the brains of my Cupid's knock'd out, and I beginne to loue, as an old man loues money, with no stomacke Lad. What haue we heere? Clo. In that you haue there. Exit A Letter. I haue sent you a daughter-in-Law, shee hath recouered the King, and vndone me: I haue wedded her, not bedded her, and sworne to make the not eternall. You shall heare I am runne away, know it before the report come. If there bee bredth enough in the world, I will hold a long distance. My duty to you. Your vnfortunate sonne, Bertram. This is not well rash and vnbridled boy, To flye the fauours of so good a King, To plucke his indignation on thy head, By the misprising of a Maide too vertuous For the contempt of Empire. Enter Clowne. Clow. O Madam, yonder is heauie newes within betweene two souldiers, and my yong Ladie La. What is the matter Clo. Nay there is some comfort in the newes, some comfort, your sonne will not be kild so soone as I thoght he would La. Why should he be kill'd? Clo. So say I Madame, if he runne away, as I heare he does, the danger is in standing too't, that's the losse of men, though it be the getting of children. Heere they come will tell you more. For my part I onely heare your sonne was run away. Enter Hellen and two Gentlemen. French E. Saue you good Madam Hel. Madam, my Lord is gone, for euer gone French G. Do not say so La. Thinke vpon patience, pray you Gentlemen, I haue felt so many quirkes of ioy and greefe, That the first face of neither on the start Can woman me vntoo't. Where is my sonne I pray you? Fren.G. Madam he's gone to serue the Duke of Florence, We met him thitherward, for thence we came: And after some dispatch in hand at Court, Thither we bend againe Hel. Looke on his Letter Madam, here's my Pasport. When thou canst get the Ring vpon my finger, which neuer shall come off, and shew mee a childe begotten of thy bodie, that I am father too, then call me husband: but in such a (then) I write a Neuer. This is a dreadfull sentence La. Brought you this Letter Gentlemen? 1.G. I Madam, and for the Contents sake are sorrie for our paines Old La. I prethee Ladie haue a better cheere, If thou engrossest, all the greefes are thine, Thou robst me of a moity: He was my sonne, But I do wash his name out of my blood, And thou art all my childe. Towards Florence is he? Fren.G. I Madam La. And to be a souldier Fren.G. Such is his noble purpose, and beleeu't The Duke will lay vpon him all the honor That good conuenience claimes La. Returne you thither Fren.E. I Madam, with the swiftest wing of speed Hel. Till I haue no wife, I haue nothing in France, 'Tis bitter La. Finde you that there? Hel. I Madame Fren.E. 'Tis but the boldnesse of his hand haply, which his heart was not consenting too Lad. Nothing in France, vntill he haue no wife: There's nothing heere that is too good for him But onely she, and she deserues a Lord That twenty such rude boyes might tend vpon, And call her hourely Mistris. Who was with him? Fren.E. A seruant onely, and a Gentleman: which I haue sometime knowne La. Parolles was it not? Fren.E. I my good Ladie, hee La. A verie tainted fellow, and full of wickednesse, My sonne corrupts a well deriued nature With his inducement Fren.E. Indeed good Ladie the fellow has a deale of that, too much, which holds him much to haue La. Y'are welcome Gentlemen, I will intreate you when you see my sonne, to tell him that his sword can neuer winne the honor that he looses: more Ile intreate you written to beare along Fren.G. We serue you Madam in that and all your worthiest affaires La. Not so, but as we change our courtesies, Will you draw neere? Enter. Hel. Till I haue no wife I haue nothing in France. Nothing in France vntill he has no wife: Thou shalt haue none Rossillion, none in France, Then hast thou all againe: poore Lord, is't I That chase thee from thy Countrie, and expose Those tender limbes of thine, to the euent Of the none-sparing warre? And is it I, That driue thee from the sportiue Court, where thou Was't shot at with faire eyes, to be the marke Of smoakie Muskets? O you leaden messengers, That ride vpon the violent speede of fire, Fly with false ayme, moue the still-peering aire That sings with piercing, do not touch my Lord: Who euer shoots at him, I set him there. Who euer charges on his forward brest I am the Caitiffe that do hold him too't, And though I kill him not, I am the cause His death was so effected: Better 'twere I met the rauine Lyon when he roar'd With sharpe constraint of hunger: better 'twere, That all the miseries which nature owes Were mine at once. No come thou home Rossillion, Whence honor but of danger winnes a scarre, As oft it looses all. I will be gone: My being heere it is, that holds thee hence, Shall I stay heere to doo't? No, no, although The ayre of Paradise did fan the house, And Angels offic'd all: I will be gone, That pittifull rumour may report my flight To consolate thine eare. Come night, end day, For with the darke (poore theefe) Ile steale away. Enter. Flourish. Enter the Duke of Florence, Rossillion, drum and trumpets, soldiers, Parrolles. Duke. The Generall of our horse thou art, and we Great in our hope, lay our best loue and credence Vpon thy promising fortune Ber. Sir it is A charge too heauy for my strength, but yet Wee'l striue to beare it for your worthy sake, To th' extreme edge of hazard Duke. Then go thou forth, And fortune play vpon thy prosperous helme As thy auspicious mistris Ber. This very day Great Mars I put my selfe into thy file, Make me but like my thoughts, and I shall proue A louer of thy drumme, hater of loue. Exeunt. omnes Enter Countesse & Steward. La. Alas! and would you take the letter of her: Might you not know she would do, as she has done, By sending me a Letter. Reade it agen. Letter. I am S[aint]. Iaques Pilgrim, thither gone: Ambitious loue hath so in me offended, That bare-foot plod I the cold ground vpon With sainted vow my faults to haue amended Write, write, that from the bloodie course of warre, My deerest Master your deare sonne, may hie, Blesse him at home in peace. Whilst I from farre, His name with zealous feruour sanctifie: His taken labours bid him me forgiue: I his despightfull Iuno sent him forth, From Courtly friends, with Camping foes to liue, Where death and danger dogges the heeles of worth. He is too good and faire for death, and mee, Whom I my selfe embrace, to set him free. Ah what sharpe stings are in her mildest words? Rynaldo, you did neuer lacke aduice so much, As letting her passe so: had I spoke with her, I could haue well diuerted her intents, Which thus she hath preuented Ste. Pardon me Madam, If I had giuen you this at ouer-night, She might haue beene ore-tane: and yet she writes Pursuite would be but vaine La. What Angell shall Blesse this vnworthy husband, he cannot thriue, Vnlesse her prayers, whom heauen delights to heare And loues to grant, repreeue him from the wrath Of greatest Iustice. Write, write Rynaldo, To this vnworthy husband of his wife, Let euerie word waigh heauie of her worth, That he does waigh too light: my greatest greefe, Though little he do feele it, set downe sharpely. Dispatch the most conuenient messenger, When haply he shall heare that she is gone, He will returne, and hope I may that shee Hearing so much, will speede her foote againe, Led hither by pure loue: which of them both Is deerest to me, I haue no skill in sence To make distinction: prouide this Messenger: My heart is heauie, and mine age is weake, Greefe would haue teares, and sorrow bids me speake. Exeunt. A Tucket afarre off. Enter old Widdow of Florence, her daughter Violenta and Mariana, with other Citizens. Widdow. Nay come, For if they do approach the Citty, We shall loose all the sight Diana. They say, the French Count has done Most honourable seruice Wid. It is reported, That he has taken their great'st Commander, And that with his owne hand he slew The Dukes brother: we haue lost our labour, They are gone a contrarie way: harke, you may know by their Trumpets Maria. Come lets returne againe, And suffice our selues with the report of it. Well Diana, take heed of this French Earle, The honor of a Maide is her name, And no Legacie is so rich As honestie Widdow. I haue told my neighbour How you haue beene solicited by a Gentleman His Companion Maria. I know that knaue, hang him, one Parolles, a filthy Officer he is in those suggestions for the young Earle, beware of them Diana; their promises, entisements, oathes, tokens, and all these engines of lust, are not the things they go vnder: many a maide hath beene seduced by them, and the miserie is example, that so terrible shewes in the wracke of maiden-hood, cannot for all that disswade succession, but that they are limed with the twigges that threatens them. I hope I neede not to aduise you further, but I hope your owne grace will keepe you where you are, though there were no further danger knowne, but the modestie which is so lost Dia. You shall not neede to feare me. Enter Hellen. Wid. I hope so: looke here comes a pilgrim, I know she will lye at my house, thither they send one another, Ile question her. God saue you pilgrim, whether are bound? Hel. To S[aint]. Iaques la grand. Where do the Palmers lodge, I do beseech you? Wid. At the S[aint]. Francis heere beside the Port Hel. Is this the way? A march afarre. Wid. I marrie ist. Harke you, they come this way: If you will tarrie holy Pilgrime But till the troopes come by, I will conduct you where you shall be lodg'd, The rather for I thinke I know your hostesse As ample as my selfe Hel. Is it your selfe? Wid. If you shall please so Pilgrime Hel. I thanke you, and will stay vpon your leisure Wid. You came I thinke from France? Hel. I did so Wid. Heere you shall see a Countriman of yours That has done worthy seruice Hel. His name I pray you? Dia. The Count Rossillion: know you such a one? Hel. But by the eare that heares most nobly of him: His face I know not Dia. What somere he is He's brauely taken heere. He stole from France As 'tis reported: for the King had married him Against his liking. Thinke you it is so? Hel. I surely meere the truth, I know his Lady Dia. There is a Gentleman that serues the Count, Reports but coursely of her Hel. What's his name? Dia. Monsieur Parrolles Hel. Oh I beleeue with him, In argument of praise, or to the worth Of the great Count himselfe, she is too meane To haue her name repeated, all her deseruing Is a reserued honestie, and that I haue not heard examin'd Dian. Alas poore Ladie, 'Tis a hard bondage to become the wife Of a detesting Lord Wid. I write good creature, wheresoere she is, Her hart waighes sadly: this yong maid might do her A shrewd turne if she pleas'd Hel. How do you meane? May be the amorous Count solicites her In the vnlawfull purpose Wid. He does indeede, And brokes with all that can in such a suite Corrupt the tender honour of a Maide: But she is arm'd for him, and keepes her guard In honestest defence. Drumme and Colours. Enter Count Rossillion, Parrolles, and the whole Armie. Mar. The goddes forbid else Wid. So, now they come: That is Anthonio the Dukes eldest sonne, That Escalus Hel. Which is the Frenchman? Dia. Hee, That with the plume, 'tis a most gallant fellow, I would he lou'd his wife: if he were honester He were much goodlier. Is't not a handsom Gentleman Hel. I like him well Di. 'Tis pitty he is not honest: yonds that same knaue That leades him to these places: were I his Ladie, I would poison that vile Rascall Hel. Which is he? Dia. That Iacke-an-apes with scarfes. Why is hee melancholly? Hel. Perchance he's hurt i'th battaile Par. Loose our drum? Well Mar. He's shrewdly vext at something. Looke he has spyed vs Wid. Marrie hang you Mar. And your curtesie, for a ring-carrier. Enter. Wid. The troope is past: Come pilgrim, I wil bring you, Where you shall host: Of inioyn'd penitents There's foure or fiue, to great S[aint]. Iaques bound, Alreadie at my house Hel. I humbly thanke you: Please it this Matron, and this gentle Maide To eate with vs to night, the charge and thanking Shall be for me, and to requite you further, I will bestow some precepts of this Virgin, Worthy the note Both. Wee'l take your offer kindly. Exeunt. Enter Count Rossillion and the Frenchmen, as at first. Cap.E. Nay good my Lord put him too't: let him haue his way Cap.G. If your Lordshippe finde him not a Hilding, hold me no more in your respect Cap.E. On my life my Lord, a bubble Ber. Do you thinke I am so farre Deceiued in him Cap.E. Beleeue it my Lord, in mine owne direct knowledge, without any malice, but to speake of him as my kinsman, hee's a most notable Coward, an infinite and endlesse Lyar, an hourely promise-breaker, the owner of no one good qualitie, worthy your Lordships entertainment Cap.G. It were fit you knew him, least reposing too farre in his vertue which he hath not, he might at some great and trustie businesse, in a maine daunger, fayle you Ber. I would I knew in what particular action to try him Cap.G. None better then to let him fetch off his drumme, which you heare him so confidently vndertake to do C.E. I with a troop of Florentines wil sodainly surprize him; such I will haue whom I am sure he knowes not from the enemie: wee will binde and hoodwinke him so, that he shall suppose no other but that he is carried into the Leager of the aduersaries, when we bring him to our owne tents: be but your Lordship present at his examination, if he do not for the promise of his life, and in the highest compulsion of base feare, offer to betray you, and deliuer all the intelligence in his power against you, and that with the diuine forfeite of his soule vpon oath, neuer trust my iudgement in anie thing Cap.G. O for the loue of laughter, let him fetch his drumme, he sayes he has a stratagem for't: when your Lordship sees the bottome of this successe in't, and to what mettle this counterfeyt lump of ours will be melted if you giue him not Iohn drummes entertainement, your inclining cannot be remoued. Heere he comes. Enter Parrolles. Cap.E. O for the loue of laughter hinder not the honor of his designe, let him fetch off his drumme in any hand Ber. How now Monsieur? This drumme sticks sorely in your disposition Cap.G. A pox on't, let it go, 'tis but a drumme Par. But a drumme: Ist but a drumme? A drum so lost. There was excellent command, to charge in with our horse vpon our owne wings, and to rend our owne souldiers Cap.G. That was not to be blam'd in the command of the seruice: it was a disaster of warre that C�sar him selfe could not haue preuented, if he had beene there to command Ber. Well, wee cannot greatly condemne our successe: some dishonor wee had in the losse of that drum, but it is not to be recouered Par. It might haue beene recouered Ber. It might, but it is not now Par. It is to be recouered, but that the merit of seruice is sildome attributed to the true and exact performer, I would haue that drumme or another, or hic iacet Ber. Why if you haue a stomacke, too't Monsieur: if you thinke your mysterie in stratagem, can bring this instrument of honour againe into his natiue quarter, be magnanimious in the enterprize and go on, I wil grace the attempt for a worthy exploit: if you speede well in it, the Duke shall both speake of it, and extend to you what further becomes his greatnesse, euen to the vtmost syllable of your worthinesse Par. By the hand of a souldier I will vndertake it Ber. But you must not now slumber in it Par. Ile about it this euening, and I will presently pen downe my dilemma's, encourage my selfe in my certaintie, put my selfe into my mortall preparation: and by midnight looke to heare further from me Ber. May I bee bold to acquaint his grace you are gone about it Par. I know not what the successe wil be my Lord, but the attempt I vow Ber. I know th'art valiant, And to the possibility of thy souldiership, Will subscribe for thee: Farewell Par. I loue not many words. Exit Cap.E. No more then a fish loues water. Is not this a strange fellow my Lord, that so confidently seemes to vndertake this businesse, which he knowes is not to be done, damnes himselfe to do, & dares better be damnd then to doo't Cap.G. You do not know him my Lord as we doe, certaine it is that he will steale himselfe into a mans fauour, and for a weeke escape a great deale of discoueries, but when you finde him out, you haue him euer after Ber. Why do you thinke he will make no deede at all of this that so seriouslie hee dooes addresse himselfe vnto? Cap.E. None in the world, but returne with an inuention, and clap vpon you two or three probable lies: but we haue almost imbost him, you shall see his fall to night; for indeede he is not for your Lordshippes respect Cap.G. Weele make you some sport with the Foxe ere we case him. He was first smoak'd by the old Lord Lafew, when his disguise and he is parted, tell me what a sprat you shall finde him, which you shall see this verie night Cap.E. I must go looke my twigges, He shall be caught Ber. Your brother he shall go along with me Cap.G. As't please your Lordship, Ile leaue you Ber. Now wil I lead you to the house, and shew you The Lasse I spoke of Cap.E. But you say she's honest Ber. That's all the fault: I spoke with hir but once, And found her wondrous cold, but I sent to her By this same Coxcombe that we haue i'th winde Tokens and Letters, which she did resend, And this is all I haue done: She's a faire creature, Will you go see her? Cap.E. With all my heart my Lord. Exeunt. Enter Hellen, and Widdow. Hel. If you misdoubt me that I am not shee, I know not how I shall assure you further, But I shall loose the grounds I worke vpon Wid. Though my estate be falne, I was well borne, Nothing acquainted with these businesses, And would not put my reputation now In any staining act Hel. Nor would I wish you. First giue me trust, the Count he is my husband, And what to your sworne counsaile I haue spoken, Is so from word to word: and then you cannot By the good ayde that I of you shall borrow, Erre in bestowing it Wid. I should beleeue you, For you haue shew'd me that which well approues Y'are great in fortune Hel. Take this purse of Gold, And let me buy your friendly helpe thus farre, Which I will ouer-pay, and pay againe When I haue found it. The Count he woes your daughter, Layes downe his wanton siedge before her beautie, Resolue to carrie her: let her in fine consent As wee'l direct her how 'tis best to beare it: Now his important blood will naught denie, That shee'l demand: a ring the Countie weares, That downward hath succeeded in his house From sonne to sonne, some foure or fiue discents, Since the first father wore it. This Ring he holds In most rich choice: yet in his idle fire, To buy his will, it would not seeme too deere, How ere repented after Wid. Now I see the bottome of your purpose Hel. You see it lawfull then, it is no more, But that your daughter ere she seemes as wonne, Desires this Ring; appoints him an encounter; In fine, deliuers me to fill the time, Her selfe most chastly absent: after To marry her, Ile adde three thousand Crownes To what is past already Wid. I haue yeelded: Instruct my daughter how she shall perseuer, That time and place with this deceite so lawfull May proue coherent. Euery night he comes With Musickes of all sorts, and songs compos'd To her vnworthinesse: It nothing steeds vs To chide him from our eeues, for he persists As if his life lay on't Hel. Why then to night Let vs assay our plot, which if it speed, Is wicked meaning in a lawfull deede; And lawfull meaning in a lawfull act, Where both not sinne, and yet a sinfull fact. But let's about it. | Summary: In twenty-three lines, Shakespeare introduces the city of Florence, Italy, to the play while that city's duke puzzles aloud to a French nobleman about the king of France's neutrality in the Italian wars. The French lord concurs: "Holy seems the quarrel / Upon your Grace's part; black and fearful / On the opposer." In Scene 2, the clown has returned to Rousillon, where he delivers a letter from Bertram to his mother advising her that he has run away from his marriage; in the letter, he says: "If there be breadth enough in the world, I will hold a long distance." This upsets the Countess: "This is not well, rash and unbridled boy, / To fly the favours of so good a King." Helena's distress, when she reads Bertram's letter to her, compounds the feeling. She labels his note a "passport" -- that is, a license to beg on the open road -- and says that it is a "dreadful sentence." When thou canst get the ring upon my finger,Which never shall come off, and show me a childBegotten of thy body that I am father to, then callMe husband; but in such a "then" I write a "Never." The Countess disavows Bertram as her son and then asks whether or not he is still traveling in the company of Parolles, the "very tainted fellow, and full of wickedness." The scene ends with a monologue by Helena, who vows to leave France to clear the way for Bertram to return home from the dangerous wars: No; come thou home, Rousillon,Whence honor but of danger wins a scar,As oft it loses all. I will be gone, -- My being here it is that holds thee hence.Shall I stay here to do it? No, no. | 6,137 | 427 | booksum | en |
Write a title and summarize: The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome. MaoP belongs to a group of proteins conserved in the Enterobacteria that coevolved with Dam methylase including SeqA, MukBEF and MatP that are all involved in the control of chromosome conformation and segregation. Analysis of DNA rings excised from the chromosome demonstrated that the single maoS site is required in cis on the chromosome to exert its effect while MaoP can act both in cis and in trans. The position of markers in the Ori region was affected by inactivating maoP. However, the MaoP/maoS system was not sufficient for positioning the Ori region at the ¼–¾ regions of the cell. We also demonstrate that the replication and the resulting expansion of bulk DNA are localized centrally in the cell. Implications of these results for chromosome positioning and segregation in E. coli are discussed. The size of genomes with respect to cellular dimensions imposes the need for extensive chromosome condensation that is compatible with the genome replication and the expression of genetic information. The bacterial chromosome is organized at multiple levels from the genome content (including the distribution of DNA motifs, genes and replication arms), to chromatin composition, supercoiling domains and large chromosomal domains [1,2]. The E. coli chromosome is divided into four large domains called macrodomains (MDs) [3,4]. MDs are defined as large regions in which DNA interactions occurred preferentially, whilst DNA interactions between different MDs are restricted. The Ori MD contains the origin of replication oriC, the opposite Ter MD contains the replication terminus and the chromosome dimer resolution dif site, whilst the Left and Right MDs flank the Ter MD. Two non-structured (NS) regions (NSRight and NSLeft) flank the Ori MD. DNA sites within the NS regions can interact with both flanking MDs [4]. MD organization influences the segregation of sister chromatids and the mobility of chromosomal DNA [5]. Each MD occupies a specific territory inside the nucleoid and is segregated with specific properties [5]. The molecular details of Ter MD organisation and segregation have previously been uncovered. The protein MatP (Macrodomain Ter protein) binds to a specific DNA sequence, termed matS, that is repeated 23 times within the 800 kb Ter region [6,7]. Furthermore, the interaction of MatP with the division apparatus associated protein ZapB promotes the anchoring of the Ter MD at mid-cell to control segregation of the Ter MD [8]. The mobility of DNA markers in MDs is highly reduced compared to NS regions. In the Ter MD, constraints on DNA mobility resulted both from the binding of MatP to matS sites and from its interaction with the divisome. The mechanisms responsible for structuring and constraining DNA mobility in other MDs have not been described. The understanding of bacterial chromosome segregation has improved considerably in recent years [1]. The Ori region from bacterial chromosomes plays a pivotal role in chromosome organization and segregation as it is replicated and segregated early in cell division and its positioning impacts the cellular organization of the chromosome in the cell [2]. In many bacterial species, ParABS play a critical role in the condensation and segregation of the Ori region. Although present on different low copy number plasmids in E. coli (e. g. F, P1) [9], ParABS or any analogous system has not been found in the chromosome of E. coli or other enteric bacteria. In E. coli, a specific cis-acting site called migS affects bipolar positioning of oriC but its deletion does not cause any severe defects in chromosome partitioning [10]. Different processes have been proposed to contribute to the partitioning and segregation of the E. coli chromosome, including the radial confinement of nucleoid DNA [11], migS-driven separation of Ori regions [10], entropic exclusion of sister chromosomes [12], differential condensation levels of the chromosomal regions [13] and a MukB-TopoIV interaction for decatenation and segregation of newly replicated Ori DNA [14]. The final destination of the Ori region is controlled and maybe responsible for the transversal organization of the chromosome in these species [15–17]. However, the mechanisms that control the positioning of the Ori region remain undefined. Genomic rearrangements have previously been used to unravel the principles of chromosome organization [18–25]. In many cases, the recombination between inverted recombining sites was used to promote the rearrangement either using general recombination systems or site specific recombination modules. Although this method can be applied with sites dispersed all over the genome it can unwittingly affect several parameters at the same time. For example, inversion of a segment encompassing the Right and Ori MD will not only generate hybrid MDs but will also affect the orientation of genes and sequences inside hybrid MDs and the intervening segment generating possible replication-transcription conflicts [26]. To limit potential replication-transcription conflicts resulting from genetic rearrangements, we previously developed a strategy that allows the transposition of any chromosomal segment at any position in the chromosome in any orientation. Thus one can change the position of any DNA segment whilst preserving its orientation relative to the replication process [27]. These rearrangements rely on a cut-and-paste process reminiscent of a transposition event and involve the site-specific recombination module of bacteriophage λ. In an attempt to define the mechanism responsible for structuring the Ori MD, this current study has generated new chromosome configurations by transposing different regions of the Ori MD, Right MD or NSRight region without affecting gene orientation. By analyzing the mobility of transposed segments we show that different regions of the Ori MD obtained new mobility properties when present in a different genomic context. Interestingly, a specific 17 bp sequence from the Ori region, termed maoS, was responsible for restricting DNA mobility in adjacent regions. A gene adjacent to maoS, termed maoP (previously known as yifE) was also required for constraining marker mobility in the Ori region. Inactivation of maoP removed constraints on DNA mobility in the Ori MD and allowed long-range interaction between Ori and Right MDs. The maoS/MaoP system contributed to the control of Ori region choreography, as inactivation of maoP affected the position of Ori markers. We also demonstrate that DNA replication and the resulting expansion of bulk DNA positions the nucleoid in the middle of the cell. Implications of these results for nucleoid positioning and chromosome conformation are discussed. Our first goal was to understand if sequences within the Ori domain influence DNA mobility. To do this, we monitored movement of several chromosomal regions. The mobility of foci was determined by measuring the distance traveled by a focus from the home position at 10 sec intervals for a period of 5 min [5]. The motion of these regions was compared before and after chromosomal segments had been displaced by transposition. The transposed (Td) segments are labeled according to their original position with a numerical suffix, i. e. segments from the Ori region are called “ori Td-1”, “ori Td-2” and etcetera. A line below the genetic map indicates the transposed segments, and an arrowhead represents the insertion site. Different segments from the Ori and Right MDs or from the NSRight region were inserted in the Right or Ori regions (Fig 1 and S1 Fig). When two segments proximal to oriC (i. e the 138-kb ori Td-1 (Fig 1A) and the 302-kb ori Td-2 (S1A Fig) ) were transposed to the Right MD at 17’, markers in the transposed Ori segment (Ori-6 and Ori-7) showed the same constraints on DNA mobility as in the original configuration (Fig 1A and S1A Fig). In this configuration, markers in the Right MD (Right-5) and in the NSRight region (NSR-5) also showed the same mobility as in the WT context. In contrast, other markers in the Ori region not involved in the transposition event (markers Ori-3 and Ori-4) displayed a higher mobility (Fig 1A). Transposition of segments distal to oriC (i. e the 318kb ori Td-4 (Fig 1B) and the 143kb ori Td-3 (S1 Fig) ) to the same locus in the Right MD resulted in transposed Ori markers (Ori-3 and Ori-4) with an increased mobility at their new location (Fig 1B and S1B Fig). The mobility of Ori markers not involved in the transposition remained unchanged. Interestingly, the marker NSR-1 in the NSRight region, that was positioned closer to oriC, had a reduced mobility compared to the WT context. Altogether, these data indicate that the Ori region is composed of heterogeneous parts that have altered properties in different genetic contexts. In conclusion determinants responsible for limiting DNA mobility are not scattered homogeneously through the Ori region. Instead they are present in the ori Td-1 segment and constraints on DNA mobility are spread to adjacent segments. The results described above suggested that constraints in the Ori region can spread to NS markers if they are brought closer to C. To test this hypothesis, a 264kb segment from the NSRight region (NSR Td-2) was transposed to two different positions (respectively 92. 7 min and 87. 7 min) in the Ori region (Fig 1C and S1C Fig, respectively). At both positions, the mobility of markers NSR-1 and NSR-2 in the transposed segment was constraint whilst the mobility of a marker at its original position (NSR-5) was not affected (Fig 1C and S1C Fig). The mobility of markers originally located in the Ori region (Ori-3 and Ori-4) were increased following transposition even though they did not acquire the mobility of a NSR marker located the same distance from oriC in the WT configuration (Fig 1C). Finally, analysis of NSR-2 and NSR-5 mobility in a configuration where a large segment from the NSRight region (NSR Td-1) is transposed within the Right MD (Fig 1B) indicated that all markers displayed the same mobility as in the WT strain. In conclusion the insertion of segments from either the Ori region or the NSRight region into the Right MD had no consequences on the mobility of loci of the Right MD. To determine the consequences of transposing a segment of the Right MD into the Ori region, we analyzed the mobility of various markers originally located in the Right and Ori MDs following chromosome rearrangement. A 154-kb segment (right Td-1) (Fig 1D) and a 293-kb segment (right Td-2) (S1D Fig) were transposed into two different positions (respectively 92. 7 min and 87. 7 min) in the Ori MD. The mobility of markers in the transposed segment from the Right MD was unchanged. Interestingly the Ori markers (Ori-3 and Ori-4) that were separated from oriC by the transposed Right MD segment Right Td-1 had increased mobility (Fig 1D). Locus Ori-1, whose position remained unchanged, had similar constraints on DNA mobility as in the WT context (Fig 1D). Similar results were obtained for markers Right-5, Ori-3 and Ori-4 after insertion of Right Td-2 at 92. 7 and 87. 6 min (S1D Fig). In conclusion, segments from the Right MD were able to impede the transmission of mobility constraints originating from the region proximal to oriC. By utilizing the ability of transposed Right MD segments to increase the mobility of Ori MD markers, we attempted to map the determinants required for constraining DNA in the Ori MD. By inserting a segment from the Right MD at different positions in the Ori region and measuring the mobility of markers on either side of the transposed segment, it was predicted that marker mobility would be affected relative to their position to DNA constraining determinants. The 293-kb right Td-2 segment was transposed at five positions in the Ori region and marker mobility was assessed at the five positions: 87,7 min (a), 86. 7 min (b), 86. 1 min (c), 85. 1 min (d) and 84. 7 min (e) (Fig 2A). Insertion of right Td-2 at positions a, b, c, and d resulted in an increased mobility of markers distal from oriC (i. e. Ori-3 and Ori-4) whereas a marker close to oriC (i. e. Ori-5) was not affected (Fig 2A). When right Td-2 was inserted at 84. 7 min (e position) the mobility of markers distal to oriC was not affected whilst the marker close to oriC had a higher mobility (Fig 2A). In conclusion, determinants responsible for constraining the Ori region were present in a region of 69-kb (coordinates 3928200 to 3998000) between positions d and e. To precisely map the position of determinants constraining DNA mobility, four deletions corresponding to non-overlapping segments varying in size between 2 kb and 17 kb were generated in the 69 kb region (Fig 2B). Two regions could not be deleted because of the presence of essential genes. The deletion of the 2 kb segment (delta 15–17; coordinates 3944500–3946500) resulted in an increased mobility of the Ori MD markers (Ori-3 and Ori-4), whilst the three remaining deletions had no substantial effect (Fig 2B). This region contains three genes: hdfR, yifE and yifB. hdfR and yifE are divergent genes separated by a 118-bp long intergenic region and yifB is convergent with yifE (Fig 2C). To further define the determinants required for conferring Ori MD properties, additional deletions were performed and the mobility of markers Ori-3, Ori-4 and NSR-2 were measured (Fig 2C). These results demonstrate that both the yifE gene and the upstream intergenic region (IR) were required to constrain the mobility of markers Ori-3 and Ori-4. It is interesting to note that deletion of hdfR had an opposite effect as the mobility of all three markers was more constrained in this context. The deletion of yifB had no effect on marker mobility. In conclusion, yifE and the upstream intergenic region (IR) are required for constraining DNA mobility in the Ori MD. To characterize the determinants required for limiting mobility of Ori markers, constructs were generated to provide the IR and yifE in trans (i. e. present on a self-replicating pSC101 plasmid derivative) in different chromosomal contexts, i. e. strains deleted for yifE and the IR, or carrying the IR at different loci. The IR region presumably contains the promoter for yifE. The presence of yifE and the upstream IR on a replicating plasmid was not able to constrain the mobility of markers in the region (Ori-3 and Ori-4) when yifE and IR were deleted from the chromosome (Fig 3A). In contrast, the presence of the IR on the chromosome, either at the WT position or at NSR-1 position, together with the replicating plasmid carrying yifE and IR restored the constrained mobility of Ori-3 and Ori-4 markers in a yifE deletion mutant (Fig 3A). Interestingly, the mobility of the NSR-2 marker was constrained when the IR was inserted in the NS region (NSR-1) in the presence of the plasmid carrying yifE. These results demonstrate that the IR exerts its constraint on DNA mobility both in its left and right, and must be present in cis, whilst yifE can act in trans or in cis, either when present on the plasmid or inserted in the chromosome. To identify the determinants required in cis, various part of the IR were tested for their ability to constrain DNA mobility of marker NSR-2 when inserted at position NSR-1. We first identified a 30 nts segment in the middle of the IR and subsequently, a 17 nts long motif with the sequence CTAATACTCCGCGCCAT was shown to limit the mobility of marker NSR-2 (S2 Fig). This motif was termed maoS (Macrodomain Ori Sequence). To confirm that maoS may act in cis to constrain mobility of DNA markers, we used a site-specific recombination system to loop out various parts of the Ori region from the chromosome and analyzed the subsequent effects on marker mobility. The system relies on the λ Int and Xis recombination genes. When the site-specific recombination attR and attL sites are present in a direct orientation, the recombinase promotes the excision of the intervening segment resulting in the formation of cells carrying a chromosome split in two parts (Fig 3B). As only one of the parts carries oriC, the cells will not be viable if essential genes are present on the non-replicated ring. The efficiency of excision varies relative to the distance separating the two att sites and can be assessed by plating for viable cells as only cells that did not sustain an excision event will be viable. Reinsertion of the excised ring does not occur at a high frequency [27]. To visualize the fate of excised rings immediately following excision and to measure the mobility of markers, a parS tag targeted by ParB-GFP was inserted on the excisable segment. The segments ori Td-1 and ori Td-3 were excised from the chromosome and the mobility of markers either present on the chromosome or on the non-replicated ring were assessed (Fig 3C and 3D). The markers located in cis with maoS (Ori-3, Ori-4, Ori-6 in the WT context, Ori-6 upon excision of ori Td-1 and Ori-4 in the context of ori Td-3) were highly constrained (Fig 3C and 3D). In contrast, the mobility of markers not in cis with maoS (Ori-3 upon ori Td-1 excision and Ori-3 upon ori Td-3 excision) was increased following excision. Combined, these results indicate that constraints in the Ori region resulted from the presence of maoS exerting its effect in cis over large distances either in the chromosome or in excised rings. In conclusion, these results identified two elements required for constraining DNA in the Ori MD: the hdfR-yifE IR acting in cis and likely acting as a target sequence and the yifE gene acting in trans likely through the encoded product. The yifE gene was named maoP (Macrodomain Ori Protein). Inactivation of MaoP/maoS generated a number of abnormal cells (including ~5% of elongated cells) with a defect in nucleoid segregation (S3 Fig). In minimal medium supplemented with casamino acids or in LB medium at 30°C, the growth rate is slightly affected in a maoP mutant and the generation time is 100 min and 55 min, respectively (compared to 85 min and 50 min in WT cells). Control of replication initiation is also affected upon MaoP inactivation (S3 Fig); in minimal media, replication initiation is delayed in maoP cells compared to wt cells (about 50% of cells with 4 copies of oriC in maoP cells compared to more than 2/3 of cells with 4 copies of oriC in wt cells). The Ori region has two properties that define it as a MD [4,5]. First, the mobility of Ori markers is lower than those of flanking NS regions and second, interactions of loci present in the Ori and Right MDs occur at a low frequency. Large intrareplichore inversions, that intermingled Ori and Right MDs, occur rarely and results in a configuration that is detrimental for growth [25]. These properties of the Ori MD were analyzed upon inactivation of the MaoP/maoS system. Firstly, the mobility of markers located in the Ori region were affected whilst markers in the NS region, the Right MD or the Ter MD exhibited a mobility similar to that observed in WT cells (Fig 4A). Secondly, DNA inversions between Ori and Right MDs were not detected or occurred at a very low frequency in WT cells (Fig 4B and Table 1). However, the percentage of recombinants in Ori-Right combinations was increased in maoP deletion mutants (Fig 4B and Table 1). This increase is specific for interactions between the Ori and Right MDs as no differences were seen for interactions between the Right and Left MDs or between the Right and Ter MDs (Table 1). Furthermore, maoP inactivation increased the size of the colonies with the inverted chromosomes with respect to inversions in a WT background (Fig 4B). Combined, the results demonstrate that MaoP specifies Ori MD properties including the constraint mobility of Ori markers and restrictions on long distance interactions with loci in the Right MD. To determine if MaoP/maoS influences chromosome choreography and Ori positioning, we analyzed the number and localization of markers as a function of cell size in WT and maoP deletion cells. maoP inactivation reduced the number of cells containing 3–4 foci and increased the number cells with two foci for markers in the Ori region (Fig 4C and S4 Fig). Newborn WT cells contain an Ori-3 marker that is rapidly duplicated and segregated to ¼ and ¾ positions of the cell. In contrast, in maoP cells, duplication of foci occurred later in larger cells and the average distance separating the two segregated foci was reduced in medium sized cells with positioning at the home position being less precise (Fig 4C). In contrast, inactivation of maoP had no effect on the number and position of markers in Right and Ter MD (S5 Fig). These results indicate that MaoP/maoS system affects the timing, positioning and separation of markers in the Ori MD. Two 150-kb regions in the Ori MD contain late splitting snap loci and the late separation of newly replicated DNA in these regions resulted from an interaction with SeqA [28,29]. Inactivation of MaoP had the opposite effect as it resulted in a slight delay in the separation of loci suggesting that Ori constraining by MaoP is independent of the late seqA-dependent segregation of snap regions (S4 Fig). The molecular bases responsible for segregation of Ori to ¼–¾ positions following replication in E. coli are uncharacterized. The excision of DNA rings from Ori region revealed that two hours after excision, either no foci were evident in ~40% of cells or the focus was localized at one pole in ~60% of cells (S1 Text and S6 Fig). These results suggest that upon excision, DNA rings accumulated at the pole and in the absence of replication were inherited linearly in the cell population. Events leading to the formation of cells with polar foci and cells without foci were visualized using time-lapse experiments (S1 Text and S6 Fig). To test whether a large segment of Ori region was required to target the Ori region at the ¼- ¾ positions, we excised large segments from the Ori region, all lacking oriC. In each case, a parS tag targeted by ParB-GFP was inserted in the excisable segment carrying oriC and nucleoid DNA was visualized by DAPI staining. DNA rings composed of 500–600 kb segment excised from the Ori region behave similarly to DNA rings of 150 kb in that they were found located at one cell pole and did not co-localize with nucleoid (Fig 5). The same outcome was obtained with segments originating from either side of oriC. Combined, these results suggest that no simple determinant was identified in the ori region to specify its positioning at the ¼-¾ of the cell. To further explore how Ori regions segregate at ¼–¾ positions of the cell, we excised three additional segments of the Ori region carrying oriC. Two extended clockwise from the region close to oriC into the Ori MD whereas the third one extended counterclockwise from oriC towards the NSLeft region (Fig 6A). Because the distance separating the two recombining sites is large (> 500 kb), the rate of excision did not exceed 50% as estimated by the number of viable colonies. Excision gave rise to two circles, one 4 Mb remnant chromosome that is not replicated and one 500–600 kb “mini-chromosome” that is replicated via oriC. Excised rings and remnant chromosomes were visualized by parS/ParB-GFP, whilst nucleoid DNA was visualized by either DAPI staining or mCherry-HupA that uniformly labels cellular DNA. Two hours after the excision, two types of cells were detected. Firstly, a number of cells displayed a pattern of parS tags and nucleoids similar to cells in which excision was not induced (Fig 6B, panel “no excision”). The number varied from experiment to experiment and presumably corresponded to cells that did not sustain an excision event. Secondly, cells contained two well separated masses of DNA, with multiple parS foci specifically localized with one DNA mass while the other mass was devoid of any parS focus (Fig 6B, panel “excision”). The presence of multiple parS tags indicates that 600-kb rings carrying oriC were able to replicate. These results suggest that, at this time of observation, multiple replicated rings remained as a single nucleoid that was separated from the 4 Mb remnant chromosome found in the other half of the cell or in the adjacent new born cell. The use of strains carrying a parS tag in the mini-chromosome and either another FROS tag on the remnant chromosome (either in Ori region or in Ter region) or carrying a MatP-mCherry fusion protein confirmed the separation of mini-chromosomes carrying oriC from the remnant chromosome (Fig 6C). The position of Ori markers suggested that rings carrying oriC separated at the edge of the nucleoid. The separation of circles carrying oriC from the 4-Mb non-replicating DNA was visualized by excising DNA segments in the presence of cephalexin to block cell division. Excised rings carrying Ori markers (indicated by arrowheads) were clearly separated from the 4-Mb non-replicating rings (Fig 6D). Movies recapitulating the steps leading to the above situation were recorded using HU-labeled nucleoid and markers located in the excised rings carrying oriC (Fig 6E). Images were taken every 15 minutes during a 4h period after recombinase induction. Seventy five minutes after excision, parS foci were separated from nucleoid DNA that was devoid of Ori markers (indicated by red arrowheads) (Fig 6E). At subsequent times (105,120 and 150 min), the number of fluorescent foci as well as the amount of DNA localized with Ori markers increased. At times 120 min and 150 min, small nucleoid became apparent around Ori markers (black arrowheads). Combined, these results suggest that the replication process and the expansion of nucleoid played a major role in chromosome positioning and segregation in the cell. This current study presents the first description of a single cis acting site, maoS, and a trans acting protein, MaoP, involved in the organization of the Ori region in E. coli. Interestingly, MaoP belongs to a group of proteins involved in DNA metabolism and chromosome organization that coevolved with Dam methylase in Enterobacteria including MatP, MukBEF, SeqA, MetJ and other proteins of unknown function [30]. MatP constrains DNA over the 800-kb long Ter MD by binding to 23 matS target sites scattered throughout the Ter MD [7]. How a single maoS site constraints DNA over a distance of several hundred of kilobases is unknown, but may involve among other hypotheses a mechanism of tracking or loop extrusion (for review, see [31]) initiated at maoS to impose constraints over the large Ori MD. MaoP lacked homology to any characterized proteins, so how it structures the Ori MD remains unknown; furthermore, we cannot exclude the involvement of other components in this process (es). The Ori MD has been identified by different approaches (FISH, long-distance genetic interactions, segregation pattern and mobility of markers) and has been shown to be preferentially targeted by retrotransposition of Ll. LtrB group II intron in slow growth conditions [32]. Probing the retrotransposition pattern of this element in a maoP mutant would reveal whether this preferential targeting is dependent of Ori constraining by MaoP. The insertion of segments originating from the Right MD impedes the effects of MaoP/maoS on distal segments. This could result from determinant (s) in the Right MD that block the process or the absence of determinant (s) in the Right MD that would be required for the process to be propagated further. As the insertion of segments from NS regions acquire Ori properties when inserted in the Ori MD, it is likely that segments from the Right MD contain determinants that antagonize the propagation of Ori properties. Further structure-function analysis is required to further characterize the factors and mechanisms constraining the Ori region and to uncover how segments from the Right MD might impede this process. DNA Rings of 150–200 kb excised from various chromosomal regions accumulated at the cell pole and remained localized in this cellular space for several generations, suggesting that they could not travel across the cellular territory occupied by the nucleoid. These observations are supported by the radial confinement of the nucleoid within the cell [11] and also suggest that 100 kb-long DNA polymers that are devoid of partition systems and unable to replicate do not mix with nucleoid DNA. The position of the excised ring at the time of excision was dependent on the original location in the genetic map. Rings excised from the Ori region were found preferentially at the old poles whereas rings from the Ter MD were found at midcell/new pole. Two models describing chromosome conformation and segregation have been proposed (for review [2,33]). These models rely either on the folding of the chromosome as a random coil-like polymer compacted by external crowding forces and entropy to act as the main driver for chromosome segregation [34], or on the presence of a folded chromosome, self adhering object with intrinsic structuring, interacting with various systems that control the conformation of different regions [8,11,13,35]. The presence of multiple replicating mini-chromosomes confined only in a small fraction of the cell is in favor of the second model. In E. coli, the nucleoid DNA does not occupy the full interior cell space whilst the space at the end contains aggregated proteins [36] and is accessible to F plasmids devoid of partition system [37] or other smaller plasmids [15,38,39]. Here, large non-replicating DNA molecules accumulated in these nucleoid free territories suggesting that an unidentified system controls central positioning of the nucleoid at midcell. In many bacteria, chromosome segregation relies on the highly conserved parABS system. In many cases, the centromere-like parS sequences are found near oriC suggesting a coordination of segregation with replication. In B. subtilis, the large Ori region including oriC and parS sequences forms a macrodomain and its 3D folding pattern plays a role in the regulation of replication initiation, chromosome organization and DNA segregation [40]. Interestingly, Enterobacteria lack parABS and no analogous system has been identified. The widespread conservation of parS across diverse bacteria suggests that Par systems evolved early in the evolution of bacterial chromosomes and were subsequently lost from the Enterobacteria. This loss may have resulted either from the acquisition of alternative segregation systems or the acquisition of new properties in DNA metabolism and chromosome management that rendered parABS dispensable. Here, to identify cis-elements required for chromosome segregation we used a strategy that, if they exist, should favor their positioning at the ¼ and ¾ positions. However, only excised segments carrying oriC localized to the center of the cell; furthermore, the replication process and the expansion of nucleoid played a major role in chromosome segregation and positioning. Interestingly, multiple copies of mini-chromosomes did not immediately segregate to other half of the cell or from each other. Instead they remained together in a growing bulk suggesting that DNA confinement may be important for driving chromosome segregation and/or that an interplay with cell cycle events and/or cellular structures are required for efficient chromosome segregation. The bacterial strains and plasmids used in this study are listed in Tables 2–4 and in S1 Table. E. coli strains were grown at 30°C in Lennox broth (rich medium), or in minimal medium A supplemented with 0. 12% casaminoacids and 0. 2% glucose. Antibiotics were added when necessary. The different transpositions targeted in the chromosome were performed as in [27]. The deletions and the insertions of specific segments were done by the one-step technique in strain DY330 and transduced into working strains as previously described [41,42]. Constructed strains were verified by PCR. Flow cytometry analyses were performed as described before [5]. Inversions tests were performed as described before, for 20 min at 36°C and for 10 min at 37°C [4]. To reduce variability of the assay in the maoP deletion background, the integrase-excisionase module was integrated in the chromosome using phage HK022-based integrative vectors [43]. The strains used for excision carried two attL and attR sites derived from the λ site-specific integration module. The two att sites were inserted in the same orientation. The induction of the collision was performed at 39°C for 20 minutes and the cells were subsequently incubated at 30°C for two hours. An aliquot of the cells was plated at the same time as the non-induced strain in order to estimate lethality. When the excised segments were about 200 kb long, the level of lethality, reflecting the amount of excision, was greater than 90%. When large segments of 500 kb—600 kb were involved, the level of excision was about 50–60%. To perform time lapses analyses, cells were spread immediately after induction on slides and analyzed under the microscope. For the cephalexin experiments, cephalexin (20 μg/ml) was added 10 minutes before the induction of the excision. Cultures were grown in minimal A medium in the presence of glucose and casaminoacids without IPTG to maintain a minimal level expression of gfp-parB present on plasmid pALA2705. For the observation of nucleoids, the cells carried a mCherry fusion upstream the hupA gene. Cells were plated on an agarose pad in the same medium and immediately observed under the microscope. For the short time lapse experiments, movies were recorded automatically on a Leica microscope. Autofocus was performed at every time point on the phase contrast image and GFP fluorescence was recorded on the plane with the best phase contrast focus. Image analysis was performed with ImageJ software using the manual tracking plugin (http: //rsb. info. nih. gov/ij/index. html). The XY co-ordinates of the two poles and of the foci were recorded manually and processed automatically with Excel (Microsoft) software. The travelled distance (given in μm) was estimated over a period of 5 minutes by adding up the absolute values of the distances for all 10 sec interval as described before [5]. The (x, y) coordinates of the foci at every time point were recorded and the distance travelled in the 10 s interval calculated. For the mobility measurements at the home position, 30 foci were analyzed (15 cells with two foci for markers). To measure the mobility of markers, the distance travelled by various foci was recorded when markers were at home position. In the growth conditions used, at home position, markers of Ori, Right MDs as well as markers from NS regions were segregated in the two halves of the cells. | Title: The MaoP/maoS Site-Specific System Organizes the Ori Region of the E. coli Chromosome into a Macrodomain Summary: The Ori region from bacterial chromosomes plays a pivotal role in chromosome organization and segregation as it is replicated and segregated early in cell division cycle and its positioning impacts the cellular organization of the chromosome in the cell. The E. coli chromosome is divided into four macrodomains (MD) defined as large regions in which DNA interactions occurred preferentially. Here we have identified a new system responsible for specifying properties to the Ori MD. This system is composed of two elements: a cis-acting target sequence called maoS and a gene of unknown function acting in trans called maoP. Remarkably, MaoP belongs to a group of proteins conserved only in Enterobacteria that coevolved with the Dam DNA methylase and that includes the MatP protein structuring the Ter macrodomain and the SeqA and MukBEF proteins involved in the control of chromosome conformation and segregation. These results reveal the presence of a dedicated set of factors required in chromosome management in enterobacteria that might compensate, at least partially, for the absence of the ParABS system involved in the condensation and/or segregation of the Ori region in most bacteria. | 8,540 | 284 | lay_plos | en |
Summarize: WANG ZHAO/AFP/Getty Images Adult children in Shanghai who do not visit their parents could get sued and have their credit scores dropped, according to a new policy. The city of Shanghai is about to make it a legal obligation for adult children to visit their parents. At a press conference last week, authorities in the Chinese city said that offspring who no longer live with their parents must "visit or send greetings often," or else face some pretty severe consequences. Parents will be able to sue their children if they don't visit, and courts can in turn order the kids to return to their parents' house or nursing home, said Luo Peixin, deputy director of Shanghai's office of legislative affairs. If children don't comply with the court ruling, they could be entered into a "credit score blacklist," after which they may face difficulties opening bank accounts and getting loans, according to Reference News, an outlet managed by state agency Xinhua News. Some Chinese media outlets even went so far as to say children who don't visit their parents were like hit-and-run drivers and people who evade subway fares, Reference News noted. Kevin Frayer via Getty Images The policy is one part of an ordinance named "Regulations Safeguarding the Interests of the Elderly in Shanghai." The new policy is part of an ordinance, the name of which can be translated to "Regulations Safeguarding the Interests of the Elderly in Shanghai." It was adopted on Jan. 29 and will go into effect on May 1. By visiting home, children fulfill a moral obligation and provide spiritual comfort to their parents, state-funded media site The Paper noted. Filial piety, or the respect toward elders, is an important part of Chinese Confucianism, a centuries-old philosophy that modern Chinese society still follows closely. Another reason behind the policy may be the fact that Shanghai is facing an aging population, and the city hopes that filial visits will help cover pension costs, Reference News noted. People over 60 accounted for about 30 percent of the city's total population and the group is expected to grow in the next few years, according to official statistics. Qilai Shen/Bloomberg via Getty Images One reason behind the policy may be the fact that Shanghai is facing an aging population, and the city hopes that filial visits will help cover pension costs. Social media users across the country took to Weibo, a popular Chinese microblogging platform, to voice their concerns about the policy. Some worried that children who had unpleasant upbringings would be mandated to visit home. "What if parents weren't good parents, and abused their children, favored males over females, or did other discriminatory things?" one user wrote. "Then are the kids still obliged to go home often? Would they have to spend money to receive more punishment?" Many others were perplexed by the government's insistence on intervening in family matters. "Even visiting home has become a law enforcement issue. Sigh," another user, named Witty Lone Observer, wrote. Whether or not children visit their parents "is a reflection of a person's upbringing," Sunzixx posted. "Why do we have to tie these things to the law?" This isn't the first time that the Chinese state has intervened in family matters. The communist country announced a one-child policy in 1979 with the aim of curbing population growth, which it feared would impede development. It then amended the law to establish a two-child policy last October, in an attempt to address the problem of an increasingly large retired population being supported by a shrunken working-age population. A public court hearing recently took place in village in rural Sichuan province, observed by nearly all the area's residents. The hearing concerned a 73-year-old women suing her four children for failure to take care of her, West China Metropolis Daily reported. The hearing took place in the city of Pengzhou on Sept. 30. For the purpose of spreading legal knowledge, the circuit court decided to hear the case on a piece of open farmland. The event attracted nearly 100 spectators. At the "court," the woman demanded to live with her eldest son and his family. She asked him to provide her with a place to stay, and to give her 30 kilograms of rice every month. She also wanted him to farm her contracted land. On top of that, she demanded that each of her four children provide her an alimony of 100 yuan per month and visit her on her birthday and festivals. During the hearing, which lasted over an hour, no consensus was reached. The woman's children agreed to provide money, but were unable to agree on who she would live with. They also disagreed about how to divide their mother's medical expenses. The judge said a ruling will be delivered on the case before the end of the month. Regardless of the verdict, the educational function of the hearing was ruled a success. “What impressed me most was that all children have a responsibility to support their elders, no matter if they inherited property from their parents. Married daughters must also be dutiful,” one spectator remarked. | Summary: It was a bizarre location for an even more bizarre court case to play out. During a hearing in China's rural Sichuan province on Sept. 30-held in a grassy field in front of 100 spectators so they could learn about the legal process-a 73-year-old woman demanded that her four children be forced to pay up for not taking care of her, reports the People's Daily. The unnamed woman asked that she be allowed to stay with her eldest son and his family and be given more than 65 pounds of rice each month, along with a monthly allowance of $15 from each of her children, whom she hoped would be required to visit her on special occasions, like her birthday. Her children agreed to the monthly payment but couldn't decide how their mother's medical expenses would be paid or which sibling she should live with. A judge's decision is expected later this month. Similar cases have been heard in China before. In fact, Shanghai adopted a policy earlier this year allowing children who don't regularly call or visit their parents to be put on a "credit score blacklist," per the Huffington Post. Officials at the time compared those children to hit-and-run drivers. A spectator at this hearing claimed to have learned "that all children have a responsibility to support their elders, no matter if they inherited property from their parents." | 1,133 | 302 | multi_news | en |
Write a title and summarize: Elon Musk, Virginia Woolf, Lady Gaga y Albert Einstein son ejemplos de genios del pasado y presente según Wright. Cada uno de ellos no estaría a la altura de los estándares académicos actuales y, aún así, todos han pasado a la historia por su genialidad en las artes o la ciencia. Incluso muchos de los genios de la actualidad -como Bill Gates, Bob Dylan o Oprah Winfrey- abandonaron sus estudios e igual alcanzaron el éxito y reconocimiento en sus respectivas áreas. "El coeficiente intelectual y las notas académicas están sobrevalorados", le dice a BBC Mundo el doctor en musicología estadounidense Craig Wright, quien lleva más de dos décadas estudiando a las personas más brillantes de la historia y el presente. Wright acaba de publicar el libro The Hidden Habits of Genius: Beyond Talent, IQ, and Grit—Unlocking the Secrets of Greatness ("Los hábitos ocultos de los genios: más allá del talento, el coeficiente intelectual y el coraje, desvelando los secretos de la grandeza"), donde detalla 14 rasgos que tienen en común los genios. Final de Quizás también te interese Resulta ser que en este listado tampoco están otras características usualmente asociadas a los logros excepcionales de la humanidad, como tener un enorme talento. La definición de genialidad varía según "a quién le preguntes y cuándo", reconoce Wright. Pero si hoy le preguntas a él te dirá que "un genio es una persona con poderes mentales extraordinarios cuyos originales trabajos o conceptos cambian a la sociedad de alguna forma significativa para bien o para mal en todas las culturas y a lo largo del tiempo". Craig Wright lleva más de dos décadas investigando la genialidad. Incluso desarrolló una "fórmula de la genialidad", que es: G=SxNxD. Esto quiere decir que la genialidad (G) es igual a cuán significativo (S) es su impacto o cambio, multiplicado por el número (N) de personas impactadas y por su duración (D) en el tiempo. En otras palabras, para Wright los mayores genios son aquellos que producen el mayor impacto en la mayor cantidad de gente y por mayor tiempo. En los años que lleva dictando el llamado "curso de genialidad" en la Universidad de Yale, Wright ha visto cómo los estudiantes fruncen el ceño al escucharle decir que la cantante pop Lady Gaga es un ejemplo contemporáneo de genialidad o, por el contrario, que el deportista con más medallas olímpicas en la historia, el nadador Michael Phelps, no lo es. También ha sido testigo de cómo la amplia mayoría de sus alumnos levanta la mano cuando en la primera clase les pregunta quiénes desearían ser genios y, luego, al finalizar el curso, solo unos pocos lo siguen queriendo. De esto y más hablamos con Wright en esta entrevista. El diccionario de la Real Academia Española define la palabra "genio" como: "Capacidad mental extraordinaria para crear o inventar cosas nuevas y admirables". ¿Qué opina al respecto? Creo que es una definición limitada. Es lo que yo llamaría del "genio en potencia", porque tiene el potencial de convertirse en un genio pero aún no lo es. Los académicos que escribieron esta definición están diciendo que todo lo que debes hacer es ser capaz de usar tu cerebro para crear ideas originales. Eso incluye que el creador se quede con la idea para sí mismo. Esto es algo que podemos debatir y para ello me gustaría invocar la imagen de un Albert Einstein solo en una isla abandonada. Estando allí a él se le podría ocurrir que E=mc², podría pensar la teoría de la relatividad general y así sucesivamente, pero no le podría comunicar sus ideas a nadie y nunca escucharíamos hablar de Albert Einstein. Si Einstein hubiese pensado sus grandes teorías estando solo en una isla desierta y no se las hubiese podido comunicar a nadie, ¿igual sería un genio? Según la definición de la academia, Einstein igual sería un genio. Según mi definición, no, porque no tendría ningún impacto en ninguna persona del mundo. Por eso esto es algo que abre una suerte de debate filosófico. ¿Cuál cree que no es la forma de definir la genialidad? Como explico en mi libro, el coeficiente intelectual está sobrevalorado. Las pruebas estandarizadas de coeficiente intelectual son una forma de medir una capacidad en particular que es mayormente hereditaria. Al estudiar estos grandes individuos a lo largo de los siglos puedes ver que eran inteligentes, pero no necesariamente hubiesen logrado un resultado súperalto -digamos, de 140 o 150- en una prueba de coeficiente intelectual. En este sentido suelo usar a los ganadores del premio Nobel como ejemplo de referencia: ha habido galardonados con coeficientes intelectuales certificados de 115, 120 o por ahí. Es decir que necesitas un coeficiente intelectual por encima del promedio para entrar en el juego. Pero luego existen otra cantidad de factores y motivaciones que son los que, a la larga, realmente impulsan a alguien hacia la grandeza y le dan la capacidad de cambiar el mundo. Lady Gaga es una persona "altamente diversificada que es creativa a lo largo de un campo amplio", escribe Wright. Justo acabo de pasar cinco días gloriosos junto a tres adolescentes de 16, 14 y casi 13 años. Ellos sacan notas muy buenas y están estudiando para estas pruebas, para poder entrar a las mejores instituciones educativas. Entonces les dije que quizás las notas no eran tan importantes, que deberían salir a explorar el mundo, hacer distintas actividades, equivocarse, caerse y tener que levantarse... Pero sus padres -mi hijo y su esposa- me dijeron que les estaba dando el mensaje equivocado, que ellos los estaban alentando para sacar buenas notas. Así que ahora los padres me ven como una influencia disruptiva (risas). Pero la verdad es que pienso que se le pone demasiada presión a los jóvenes hoy en día, si el criterio que usamos para medir su excelencia como personas es algo desacertado. ¿Entonces qué deberían hacer las personas para criar un hijo genio o convertirse en uno? Creo que lo más importante es el esfuerzo, pero ¿qué lleva a trabajar duro? Porque en verdad el esfuerzo no es un motor en sí mismo, sino la manifestación externa de otras motivaciones internas. “Si supieran cuánto trabajo costó, no le llamarían genialidad”, decía el artista Miguel Ángel, citado por Wright. La pasión es un motor que se manifiesta como trabajo duro y que puede ir desde el amor hacia algo hasta la obsesión. Así que diría que es importante incentivar las pasiones. Lo otro que noté en muchas de estas grandes mentes es que son eruditos, que saben de distintas áreas. En uno de los capítulos de mi libro hablo de la fábula del zorro y el erizo: el zorro sabe mucho de diferentes cosas y el erizo sabe mucho de una cosa sola. Entonces, ¿qué tipo de persona eres: alguien que abarca mil millas o que profundiza mil millas? La mayoría de estas personas, de una forma u otra, lo que tienen es pensamiento lateral. Ven distintas cosas de forma simultánea porque han vivido una cantidad de experiencias y, como resultado, pueden combinar distintos elementos que otros no podrían porque son aparentemente disímiles. Por eso, si uno está criando niños, es importante exponerlos a diferentes experiencias: si les gusta la ciencia, puedes incentivarlos a leer novelas; si les interesa la política, quizás pueden aprender a pintar. Los padres que fuerzan a sus hijos a centrarse en una actividad para ser el mejor nadador olímpico o el próximo nobel de Física se están equivocando. Jack Ma, el fundador de Alibaba y hombre más rico de China, dio el examen nacional ("gaokao") y logró 19 de 120 puntos en matemáticas en su segundo intento. No vamos a saber cuál es su pasión a menos que vivan distintas experiencias. Y, como se suele decir, si amas lo que haces, no tendrás que trabajar ni un solo día de tu vida. ¿Quiénes son ejemplos de genios contemporáneos según su definición y cuáles suelen sorprender más a sus estudiantes? Hay varios, pero Elon Musk es el arquetipo de genio que hace cosas aparentemente locas y en una diversidad de áreas con The Boring Company, Hyperloop, SolarCity, Tesla, SpaceX... Es el máximo ejemplo del erudito haciendo una revolución en una cantidad de aspectos distintos. Pero ¿quiénes son algunos de los genios inesperados? Si menciono, por ejemplo, a Kanye West, a Lady Gaga o a Dolly Parton, alguien va a sacudir la cabeza en negación y a preguntarme si estoy bromeando. Dolly Parton es un caso muy interesante porque es una persona muy inteligente que, irónicamente, adoptó la imagen de la "rubia tonta". Es una especie de caballo de Troya. Ella ha construido su propio imperio y sigue haciéndolo. Es un modelo a seguir de cómo debería operar una mujer de negocios en el mundo del entretenimiento hoy en día. Y, al mismo tiempo, nos está deleitando al escribir cierto tipo de canciones y llevando el sonido country a las corrientes más populares de la música. Para Wright, la cantante de música country Dolly Parton es un ejemplo de genialidad como artista y mujer de negocios. Entonces es posible decir que Dolly Parton tiene una enorme importancia, particularmente para las mujeres blancas pobres de Estados Unidos, y que su impacto es significativo respecto a la cantidad de gente que escucha su música. Aún resta ver cuánto durará todo esto. Después hay casos como el de Michael Phelps. O sea, alguien en Francia reinstituyó los Juegos Olímpicos de la antigua Grecia y simplemente dijo qué deportes incluiría y uno de ellos fue la natación. Entonces se estableció que se competiría en estilo libre, espalda, pecho y mariposa, en 100 metros, 200 metros y todo eso. Michael se mete en la piscina y resulta ser que va y viene por el mismo carril mucho más rápido que todo el resto. Pero alguien va a lograr ir más rápido que él, porque hay una selección de los tipos de cuerpo que se precisa, porque la nutrición va mejorando y quizás el entrenamiento también mejore. Pero en esto sí tiene razón la academia (española): aquí no hay un componente intelectual. Es casi como un hámster en una jaula yendo más rápido y más rápido. Me gusta pensar que ser un genio es más que ser un hámster en una jaula o un nadador en una piscina. ¿Por qué cree que la mayoría de sus estudiantes de Yale, luego de estudiar a estos genios, ya no quieren ser como ellos? El problema con estas grandes mentes es que a menudo son muy destructivos con aquellos a su alrededor porque son tan apasionados que se vuelven obsesivos. Lo único en lo cual pueden enfocarse es en lograr su propio objetivo mental porque creen que van a cambiar al mundo. Piensan que deben arreglar algo y que solo ellos lo pueden hacer. "The Hidden Habits of Genius", de la editorial HarperCollins, saldrá en español a fines de este año. Son personas muy ambiciosas que se ponen mucha presión a sí mismos y, a veces, a otros. Se pueden volver muy demandantes con los demás y subestimarlos Entonces puede no ser agradable trabajar para ellos. Son cosas que se puede escuchar sobre Jeff Bezos, Elon Musk o Bill Gates, por ejemplo. Entonces, creo que la mayoría de las personas, a medida que avanzan en el curso de genialidad, se preguntan: "¿De verdad quiero ser así? ¿Quiero cambiar el mundo para una enorme cantidad de gente o quiero que el ambiente humano a mi alrededor sea mejor para todos?". Y ese es en verdad el tema más profundo detrás de su libro también... El pequeño y sucio secreto de mi libro es que la mayoría de nosotros no vamos a cambiar al mundo de ninguna forma significativa. No obstante, aprender sobre lo que estas personas hicieron nos lleva a pensar en cosas más importantes en las que todos podemos actuar: cómo vivir nuestras vidas en relación a otras personas, cómo ser más productivos o más creativos. Porque los genios ya están obsesionados con lo que hacen y despegarán de todos modos. Mientras tanto, el resto de nosotros tenemos la oportunidad de pensar cómo queremos vivir y ajustar nuestras vidas en función de eso. Los 14 hábitos o rasgos de personalidad de los genios, según Craig Wright 1. Ética de trabajo 2. Resiliencia 3. Originalidad 4. Imaginación como la de un niño 5. Curiosidad insaciable 6. Pasión 7. Inadaptación creativa 8. Rebeldía 9. Pensamiento que atraviesa fronteras (o ser como el zorro) 10. Acción contraria o pensar lo opuesto 11. Preparación 12. Obsesión 13. Relajación 14. Concentración Ahora puedes recibir notificaciones de BBC Mundo. Descarga la nueva versión de nuestra app y actívalas para no perderte nuestro mejor contenido. | Title: Cuáles son los "hábitos ocultos de los genios" según el profesor que enseña el "curso de genialidad" en Yale Summary: Ludwig van Beethoven tenía problemas para sumar y nunca aprendió a multiplicar o dividir. Pablo Picasso no sabía el abecedario, Walt Disney se quedaba dormido en clase y a Virgina Woolf ni siquiera le permitieron ir a la escuela, a pesar de que a sus hermanos los mandaron a Cambridge. A Charles Darwin le iba tan mal en los estudios que su padre llegó a decir que sería una vergüenza para la familia y Albert Einstein se graduó en física cuarto en su generación... de un total de cinco alumnos. | 2,773 | 144 | xlsum_es | es |
Summarize: V. Stiviano says Donald Sterling's racist comments on an audio recording leaked to the public were not the first by the Los Angeles Clippers owner in conversations with her. FILE - In this Dec. 19, 2010, file photo, Los Angeles Clippers owner Donald Sterling, third right, sits with V. Stiviano, left, as they watch the Clippers play the Los Angeles Lakers during an NBA preseason... (Associated Press) "There's been a number of occasions where Mr. Sterling and I had conversations just like this one. This was one of very many," Stiviano told Barbara Walters on ABC's "20/20" in an interview that aired Friday night. "Part of what the world heard was only 15 minutes. There's a number of other hours that the world doesn't know." Sterling told Stiviano in the recording that she should not post online photos of herself with black people, including basketball great Magic Johnson, or bring black people to Clippers' games. The recording, which an attorney for Stiviano said was leaked by a third party, led to public outcry across the country and the NBA. Some sponsors dropped the Clippers and others re-evaluated their relationship with the NBA. On Tuesday, NBA Commissioner Adam Silver banned Sterling for life, fined him $2.5 million and urged league owners to force him to sell the team. A three-quarters vote by the NBA owners is required to force him to sell. Though Stiviano told Walters that Sterling should "absolutely" apologize, she said she still loves him like a father figure and does not believe he is a racist. "I think Mr. Sterling is from a different generation than I am," she said. "I think he was brought up to believe these things... segregation, whites and blacks. But through his actions he's shown that he's not a racist. He's shown to be a very generous and kind man." Stiviano said that since the ban, Sterling has felt confused, alone and not supported by those around him. "I think he's highly more traumatized and hurt by the things that he said himself," she said. "I think he can't even believe or understand sometimes the thing he says, and I think he's hurt by it. He's hurting right now." A real-estate mogul, Sterling has been accused of racial missteps before. The billionaire had paid a $2.76 million settlement to resolve a federal lawsuit accusing him of systematically excluding blacks and Hispanics from his rental properties. He also won a wrongful termination lawsuit by general manager Elgin Baylor, who accused him of various slurs and slights. Los Angeles Clippers owner Donald Sterling’s confidante V. Stiviano today told ABC’s Barbara Walters that she thinks the embattled NBA basketball owner should apologize for his racist remarks. Interested in? Add as an interest to stay up to date on the latest news, video, and analysis from ABC News. Add Interest "Yes. Absolutely,” Stiviano, 31, said when Walters asked her during an exclusive television interview in Los Angeles. "I think he’s highly more traumatized and hurt by the things that he said himself,” she added. “I think he can’t even believe or understand sometimes the thing he says, and I think he’s hurt by it. He’s hurting right now." Donald Sterling's 2002 Battle With an Ex-Mistress Has Similarities to Today NBA Bans Clippers Owner Donald Sterling For Life NBA Owner Believes Donald Sterling Ouster Vote Will Be Unanimous Why Woman Accused of Leaking Racist Rant Changed Her Name ESPN: Full Coverage Asked if she thought he would go through with a public apology, Stiviano replied: “God only knows.” This is the first television interview for Stiviano since the NBA handed Sterling a lifetime ban and a $2.5 million fine on Tuesday for his racial remarks about African-Americans featured in a leaked audiotape recording. Prior to this interview with ABC News, Stiviano had been seen in public wearing a large visor over her face, which she said made it “easier to mask the pain." The interview comes a week after TMZ released the audio recordings, which subsequently launched the league investigation that led to NBA Commissioner Adam Silver to ban Sterling for life. The NBA Board of Governors will vote on whether Sterling will be forced to sell the team. One of the mysteries surrounding the situation is how the audio recordings came to light. Stiviano, who was on the receiving end of Sterling’s rant on the leaked tapes and describes herself as black and Mexican, told Walters she did record their conversations, and shared them with friends. Stiviano said a friend leaked the audio. But, she said, this rant -- in which Sterling told Stiviano not to “promote” her relationship with black people or “bring them to my games,” -- "was not the first time” they had these conversations. “There’s been a number of occasions where Mr. Sterling and I had conversations just like this one. This was one of very many,” she said. “Part of what the world heard was only 15 minutes. There’s a number of other hours that the world doesn’t know.” Stiviano denied having a romantic relationship with Sterling, saying she is his personal assistant and sees Sterling as “a father figure” -- calling herself his "silly rabbit." She claims that people around Sterling would say negative things about her to him, saying they “poison his mind and heart about things about me,” which would drive him to say certain things. Despite the controversy around his comments, she said he is not a racist. “I think Mr. Sterling is from a different generation than I am. I think he was brought up to believe these things … segregation, whites and blacks,” Stiviano said. “But through his actions he’s shown that he’s not a racist. He’s shown to be a very generous and kind man.” Sterling, 80, has owned the Clippers since 1981. Sources also told ESPN Thursday that Sterling is battling cancer. Stiviano is also currently tangled in a legal battle with Sterling’s estranged wife Rochelle Sterling. | Summary: Well, not everybody thinks LA Clippers owner Donald Sterling is a racist: V. Stiviano, the woman who started this whole mess by recording her private conversations with him, offered up some decent words about him last night to Barbara Walters of ABC News: On racism: "I think Mr. Sterling is from a different generation than I am. I think he was brought up to believe these things... segregation, whites, and blacks. But through his actions he's shown that he's not a racist. He's shown to be a very generous and kind man." Still, Stiviano said he "absolutely" should apologize for his remarks about black people, notes AP. On their relationship: Stiviano says she is his personal assistant and never had a romantic relationship with the 80-year-old. She says she loves him as a "father figure." 'He's hurting': "I think he's highly more traumatized and hurt by the things that he said himself. I think he can't even believe or understand sometimes the thing he says, and I think he's hurt by it. He's hurting right now." On how the tapes surfaced: Stiviano said she gave the tapes to friends for safekeeping and one of them leaked the audio to the media. | 1,382 | 281 | multi_news | en |
Summarize: (CNN) -- For many fliers Sunday, it was holiday travel woes, round two. As of 6:30 p.m. ET, more than 3,000 U.S. flights were delayed and more than 190 were canceled, according to FlightAware.com. The line for security screening at Chicago Midway International Airport on Sunday morning was so long that reporter Denise Whitaker of CNN affiliate KOMO measured it: 1.2 miles. Traveler Sarah Crowder called it the worst she's seen, prompting the Chicago Tribune's Brian Cassella to call on the Transportation Security Administration to "do better." Sunday is one of the busiest travel days of the year, as millions of people return home after the Thanksgiving holiday. "This happens sometimes," Chicago Aviation Department spokeswoman Karen Pride said, according to CNN affiliate WLS. "There was a period of time earlier this morning, between 6 a.m. and 8 a.m., that lines were long because that is when most people are traveling for the holiday period." On Wednesday, the weather was widely blamed, as snow snarled traffic for parts of the country. But conditions were much better Sunday in many areas, with "warmer and more tranquil conditions" in parts of the country including the East Coast, CNN meteorologist Todd Borek said. The temperatures were in the upper 40s in Chicago. While a lot of attention goes to the airports -- where the interconnected flight travel system can have a domino effect -- the vast majority of Thanksgiving travelers hit the road. Ninety percent of travelers were expected to go by road, AAA said, helped by the lowest gas prices in five years. In all, more than 46 million people were expected to travel 50 miles or more for Thanksgiving, marking the highest volume since 2007. 10 tips for Thanksgiving travel "The average distance traveled this Thanksgiving will be 549 miles roundtrip and Americans will spend an average of $573 during the holiday weekend," AAA predicted. How's your holiday travel going? Join the conversation on Facebook. Security line at Midway Airport Sunday morning was reportedly a mile long Travelers were greeted by a long line to the security checkpoint at Midway Airport Sunday morning. Photo courtesy of Paige Southard Emma Cronin arrived at Midway Airport Sunday morning an hour before her 9:50 a.m. flight to Massachusetts. For Cronin, showing up an hour early was usual business; the 21-year-old flies from Midway Airport about five times a year. “I fly Southwest a lot,” Cronin said. “Getting through the lines usually takes 20 minutes.” She was floored to see a line that was reportedly more than a mile long. “The line continued all the way through the airport to the CTA Orange Line that goes to and from Midway,” Cronin said. “I was very nervous about missing my flight.” Sunday is the busiest day of the Thanksgiving travel period, which started two days before Thanksgiving and ends Tuesday, officials said. Like many travelers, Cronin was visiting her family in Chicago for the holiday weekend. “This happens sometimes,” Chicago Aviation Department spokeswoman Karen Pride said. “There was a period of time earlier this morning, between 6 a.m. and 8 a.m., that lines were long because that is when most people are traveling for the holiday period.” According to Pride, the lines were cleared by 9:15 a.m. “I was very surprised. The TSA people were helpful and the line moved very fast,” Cronin said. “I was really nervous about missing my flight, but I arrived at my gate and took off on time.” Security line at Midway Airport is the worst I've ever seen. pic.twitter.com/dvT5Lq7B9X — sarah crowder (@sarcro) November 30, 2014 The line for TSA at Midway airport leaves the terminal, goes to city train stop, turns around and goes back in. pic.twitter.com/E56vLi7guL — TumbleDry (@TumbleDry) November 30, 2014 | Summary: If you're reading this while you wait in line at Chicago's Midway Airport, we wish you the best of luck. Lines at security have reportedly reached more than a mile long today, Gawker reports. According to one tweet: "The line for TSA at Midway airport leaves the terminal, goes to city train stop, turns around and goes back in"; a flier confirmed as much to the Chicago Sun-Times. Another tweet notes that some people have started paying each other for better spots. A Chicago aviation official says things were bad between 6 am and 8 am, but things had cleared up by 9:15. And despite the post-Thanksgiving line length, the same passenger tells the Sun-Times that "the TSA people were helpful and the line moved very fast." Travel troubles weren't limited to lines: By mid-afternoon, some 1,800 flights had been delayed today across the US, CNN reports, with 150 canceled. | 922 | 218 | multi_news | en |
Summarize: Opinion on Financial Statements The financial statements including the accompanying notes present fairly, in all material respects, in accordance with generally accepted accounting principles, the Resolution Trust Corporation’s assets, liabilities, and equity; revenues, expenses, and accumulated deficit; and cash flows. However, misstatements may nevertheless occur in other RTC-related financial information as a result of the internal control weakness described below. Opinion on RTC Management’s Assertion About the Effectiveness of Internal Controls We evaluated RTC management’s assertion about the effectiveness of its internal controls designed to safeguard assets against loss from unauthorized acquisition, use, or assure the execution of transactions in accordance with management’s authority and with laws and regulations that have a direct and material effect on the financial statements; and properly record, process, and summarize transactions to permit the preparation of reliable financial statements and to maintain accountability for assets. RTC management fairly stated that those controls in place on December 31, 1995, provided reasonable assurance that losses, noncompliance, or misstatements material in relation to the financial statements would be prevented or detected on a timely basis. RTC management made this assertion, which is included in appendix II, based upon criteria established under the Federal Managers’ Financial Integrity Act of 1982 (FMFIA). RTC management, in making its assertion, recognized the need to improve internal controls. Our work also identified the need to improve internal controls, as described in the following section. The weakness in internal controls, although not considered a material weakness, represents a significant deficiency in the design or operation of internal controls which could have adversely affected RTC’s ability to fully meet the internal control objectives listed above. Reportable Condition RTC acted during 1995 to resolve the reportable condition related to the weaknesses in general controls over some computerized information systems identified in our audit of its 1994 financial statements. However, as reported by RTC, many of those corrective actions were not completed until late in 1995. In addition, our audit of RTC’s 1995 financial statements identified additional weaknesses related to general controls over its computerized systems such that this reportable condition continued to exist. Because RTC relied on its computerized information systems extensively, both in its daily operations and in processing and reporting financial information, the effectiveness of general controls is a significant factor in ensuring the integrity and reliability of financial data. Because corrective actions for many of the general control weaknesses identified in our 1995 and 1994 audits were not implemented until late 1995 and early 1996, our audit found that general controls still did not provide adequate assurance that some of RTC data files and computer programs were fully protected from unauthorized access and modification. In response to the weaknesses we identified, RTC and FDIC developed action plans to address the weaknesses. Prior to the completion of our audit work on June 7, 1996, FDIC reported that most of the corrective actions had been implemented, with those remaining scheduled for implementation by September 30, 1996. We plan to evaluate the effectiveness of the corrective actions as part of our 1996 audit of FDIC. During 1995, RTC performed accounting and control procedures, such as reconciliations and manual comparisons, which would have detected material data integrity problems resulting from inadequate general controls. Without these procedures, weaknesses in the general controls would raise significant concern over the integrity of the information obtained from the affected systems. Other less significant matters involving the internal control structure and its operation noted during our audit will be communicated separately to FDIC’s management, which assumed responsibility for RTC’s remaining assets and liabilities since RTC’s termination on December 31, 1995. Compliance With Laws and Regulations Our tests for compliance with selected provisions of laws and regulations disclosed no instances of noncompliance that would be reportable under generally accepted government auditing standards. However, the objective of our audit was not to provide an opinion on the overall compliance with laws and regulations. Accordingly, we do not express such an opinion. The Savings and Loan Crisis: Historical Perspective and Fiscal Implications With the termination of RTC’s operations on December 31, 1995, a significant phase of the savings and loan crisis has ended. The following sections present an historical perspective on the savings and loan crisis and RTC’s role in resolving the crisis. Specifically, the information describes (1) background on the savings and loan crisis and the creation of RTC, (2) the completion of RTC’s mission, (3) RTC’s estimated costs and funding, (4) RTC’s controls over contracting, (5) the cost of resolving the savings and loan crisis, and (6) remaining fiscal implications of the crisis. The Savings and Loan Crisis and RTC During the 1980s, the savings and loan industry experienced severe financial losses because extremely high interest rates caused institutions to pay high rates on deposits and other funds while earning low yields on their long-term loan portfolios. During this period, regulators reduced capital standards and allowed the use of alternative accounting procedures to increase reported capital levels. While these conditions were occurring, institutions were allowed to diversify their investments into potentially more profitable, but risky, activities. The profitability of many of these activities depended heavily on continued inflation in real estate values to make them economically viable. In many cases, diversification was accompanied by inadequate internal controls and noncompliance with laws and regulations, thus further increasing the risk of these activities. As a result of these factors, many institutions experienced substantial losses on their loans and investments, a condition that was made worse by an economic downturn. Faced with increasing losses, the industry’s insurance fund, the Federal Savings and Loan Insurance Corporation (FSLIC), began incurring losses in 1984. By the end of 1987, 505 savings and loan institutions were insolvent. The industry’s deteriorating financial condition overwhelmed the insurance fund which only 7 years earlier reported insurance reserves of $6.5 billion. In 1987, the Congress responded by creating the Financing Corporation (FICO) to provide financing to the FSLIC through the issuance of bonds. Through August 8, 1989, FICO provided $7.5 billion in financing to the FSLIC; however, the insurance fund required far greater funding to deal with the industry’s problems. In response to the worsening savings and loan crisis, the Congress enacted the Financial Institutions Reform, Recovery, and Enforcement Act of 1989 (FIRREA) on August 9, 1989. FIRREA abolished FSLIC and transferred its assets, liabilities, and operations to the newly-created FSLIC Resolution Fund (FRF) to be administered by the FDIC. In addition, FIRREA created a new insurance fund, the Savings Association Insurance Fund (SAIF). FIRREA also created the RTC to resolve all troubled institutions placed into conservatorship or receivership from January 1, 1989, through June 30, 1995. RTC’s overall responsibilities included managing and disposing of receivership assets and recovering taxpayer funds. In 1993, the Resolution Trust Corporation Completion Act required RTC to cease its operations on or before December 31, 1995, and transfer any remaining assets and liabilities to the FSLIC Resolution Fund. FIRREA provided RTC with a total of $50 billion in funding to resolve failed institutions and pay related expenses. FIRREA also established the Resolution Funding Corporation (REFCORP) to provide RTC with $30 billion of the $50 billion in funding through the issuance of bonds. However, funding provided to RTC by FIRREA was not sufficient and the Congress enacted subsequent legislation resulting in a total of $105 billion being made available to RTC to cover losses associated with resolutions. RTC’s Mission Substantially Completed RTC closed 747 institutions with $402 billion in book value of assets when they entered the conservatorship phase. During conservatorship, assets were reduced by $162 billion to $240 billion through sales, collections, and other adjustments. In the receivership phase, assets were further reduced by $232 billion. Thus, at December 31, 1995, RTC assets in liquidation totaled approximately $8 billion. The remaining assets were transferred to the FSLIC Resolution Fund effective January 1, 1996. RTC also fulfilled the government’s pledge to insured depositors by protecting 25 million depositor accounts. Of the $277 billion in liabilities at resolution, approximately $221 billion represented liabilities to depositors. At resolution, RTC generally transferred the deposit liabilities, along with the required funding, to one or more healthy acquiring institutions. During the receivership phase, RTC used asset recoveries to pay the remaining creditors, and to recover a portion of the amount it advanced to cover deposit liabilities. Another important part of RTC’s activities included ensuring that as many thrift violators as possible were brought to justice and that funds were recovered on behalf of taxpayers. RTC investigated, initiated civil litigation, and made criminal referrals in cases involving former officers, directors, professionals, and others who played a role in the demise of failed institutions. Approximately $2.4 billion was recovered from professional liability claims, and $26 million was collected in criminal restitution. RTC’s Estimated Costs and Funding As of December 31, 1995, RTC estimated that the total cost for resolving the 747 failed institutions was $87.9 billion. These costs represent the difference between recoveries from receivership assets and the amounts advanced to pay depositors and other creditors of failed institutions plus the expenses associated with resolving institutions. As shown in table 1, $81.3 billion, or 92 percent, of RTC’s total estimated costs have already been realized through December 31, 1995, and therefore, are known. The estimated $6.6 billion remaining at December 31, 1995, represents expected future losses on remaining receivership and corporate assets. The ultimate recoveries on those assets are subject to uncertainties. Losses of $72.2 billion were realized while institutions were in receivership and after termination. Receivership losses were realized when amounts realized from asset sales were not sufficient to repay the amounts advanced by RTC. For those institutions that were terminated, RTC realized further losses if it later sold assets for less than the price it paid when it purchased the assets from the receiverships at termination. RTC borrowed working capital funds from the Federal Financing Bank (FFB) to provide funding for insured deposits and to replace high-cost borrowing of the failed institutions. In general, these funds were expected to be repaid with the proceeds from receivership asset sales, with any shortfall being covered by loss funding. Through December 31, 1995, RTC incurred $10.2 billion in interest expense on amounts borrowed from the FFB for working capital. RTC’s administrative expenses represent overhead expenses not otherwise charged or billed back to receiverships. The portion of expenses billed back to receiverships is not included in RTC’s administrative expense total, but is included in the loss from receiverships. In addition, receiverships pay many other expenses directly which are also included in the losses from receiverships. The estimated $6.6 billion of future costs include expected losses from receiverships and terminations as well as estimated future administrative expenses. In total, the Congress provided funding to cover $105 billion of losses and expenses associated with RTC’s resolution of failed institutions. As shown in table 2, after reducing the $105 billion available for RTC’s estimated losses of $87.9 billion, an estimated $17.1 billion in unused loss funds will remain. The final amount of unused loss funds will not be known with certainty until all remaining assets and liabilities are liquidated. Loss funds not used for RTC resolution activity are available until December 31, 1997, for losses incurred by the SAIF, if the conditions set forth in the Resolution Trust Corporation Completion Act are met. Also, according to the act, unused loss funds will be returned to the general fund of the Treasury. Controls Over Contracting May Have Affected Receivership Recoveries RTC used thousands of private contractors to manage and dispose of assets from failed thrifts, including activities such as collecting income and paying expenses. The estimated recoveries from receiverships included in RTC’s financial statements include the receipts collected and disbursements made by contractors that perform services for receiverships. As we previously reported, weak operating controls over contract issuance and contractor oversight may have affected the amounts RTC ultimately recovered from its receiverships. While we assess, as part of our financial statement audit, internal accounting controls over receivership receipts and disbursements, RTC’s operating controls over contract issuance and contractor oversight are not part of the scope of our audit. These operating controls were reviewed by RTC’s Inspector General and Office of Contract Oversight and Surveillance, as well as by GAO in other reviews. RTC took various actions to improve the process of contract issuance and contractor oversight, and had placed increased emphasis on the process of closing out contracts to ensure that contractors have fulfilled all contractual responsibilities. However, results of audits conducted by RTC’s Inspector General and Office of Contract Oversight and Surveillance demonstrated that despite RTC’s actions to correct contracting problems, the effects of early neglect of contracting operations remained. These audits identified internal control problems with RTC’s auction contracts and with RTC’s general oversight of contractors. These audits also identified significant performance problems with contracts that were issued before many contracting reforms and improvements were implemented by RTC. During 1995, RTC closed many contracts, pursued contract audit resolution, identified contracts necessary to accomplish the remaining workload after RTC’s termination, and processed contract modifications to transfer them to FDIC. However, estimated future recoveries from RTC receiverships remain vulnerable to the risks associated with early weaknesses in contractor oversight and performance. As a result of these operating weaknesses, RTC could not be sure that it has recovered all it should have recovered from its receiverships. RTC’s Costs Represent Only a Portion of the Total Cost of the Savings and Loan Crisis RTC’s costs for its responsibilities in resolving the savings and loan crisis represent only a portion of the total costs of the savings and loan crisis. The cost associated with FSLIC assistance and resolutions represents another sizable direct cost. In addition, the total cost includes indirect costs related to tax benefits granted in FSLIC assistance agreements. Of the $160.1 billion in total direct and indirect costs, approximately $132.1 billion, or 83 percent was provided from taxpayer funding sources. The remaining $28.0 billion, or 17 percent was provided from industry assessments and other private sources. (See Figure 1.) Direct Costs As shown in table 3, the direct costs associated with resolving the savings and loans crisis include the cost of RTC resolutions, FSLIC activity, and supervisory goodwill claims. All of the funding for the estimated $152.6 billion in estimated costs related to FSLIC and RTC has been provided as of December 31, 1995. However, the cost of the claims is currently uncertain. Resolution Trust Corporation RTC resolved 747 failed institutions through June 30, 1995, when its authority to close failed thrifts expired. As of December 31, 1995, the total estimated losses associated with RTC’s resolved institutions is $87.9 billion. Taxpayer funding for RTC’s direct costs is estimated to be $81.9 billion, which is made up of $56.6 billion in appropriations and $25.3 billion related to the government’s responsibility attributable to the REFCORP transaction. The private sources of funding for RTC activity totaled $6.0 billion, consisting of $1.2 billion contributed to RTC from the Federal Home Loan Banks, and $4.8 billion from SAIF and the Federal Home Loan Banks to support the REFCORP transaction. FSLIC Costs As of December 31, 1995, the total estimated costs associated with FSLIC activity was $64.7 billion. The estimated cost includes expenses and liabilities arising from FSLIC assistance provided to acquirers of failed or failing savings and loan institutions and FSLIC resolution activity since January 1, 1986. Taxpayer funding for FSLIC’s costs consists of appropriations used by the FSLIC Resolution Fund and totaled $42.7 billion. The private sources of funding for the FSLIC costs include $13.8 billion from FSLIC capital and industry assessments and $8.2 billion provided by FICO. Supervisory Goodwill Claims An additional cost of the savings and loan crisis results from the federal government’s legal exposure related to supervisory goodwill and other forbearances from regulatory capital requirements granted to the acquirers of troubled savings and loan institutions in the 1980s. As of December 31, 1995, there were approximately 120 pending lawsuits which stem from legislation that resulted in the elimination of supervisory goodwill and other forbearances from regulatory capital. These lawsuits assert various legal claims including breach of contract or an uncompensated taking of property resulting from the FIRREA provisions regarding minimum capital requirements for thrifts and limitations as to the use of supervisory goodwill to meet minimum capital requirements. One case has resulted in a final judgment of $6 million against FDIC, which was paid by FRF. On July 1, 1996, the United States Supreme Court concluded that the government is liable for damages in three other cases in which the changes in regulatory treament required by FIRREA led the government to not honor its contractual obligations. However, because the lower courts had not determined the appropriate measure or amount of damages, the Supreme Court returned the cases to the Court of Federal Claims for further proceedings. Until the amounts of damages are determined by the court, the amount of additional cost from these three cases is uncertain. Further, with respect to the other pending cases, the outcome of each case and the amount of any possible damages will depend on the facts and circumstances, including the wording of agreements between thrift regulators and acquirers of troubled savings and loan institutions. Estimates of possible damages suggest that the additional costs associated with these claims may be in the billions. The Congressional Budget Office’s December 1995 update of its baseline budget projections increased its projection of future federal outlays for fiscal years 1997 through 2002 by $9 billion for possible payments of such claims. Indirect Costs As shown in table 3, the estimated cost of special tax benefits related to FSLIC assistance agreements represents an indirect cost of the savings and loan crisis. The estimated total cost for these tax benefits is $7.5 billion, which will be funded using taxpayer sources. Acquiring institutions received various tax benefits associated with FSLIC assistance agreements. For instance, for tax purposes, assistance paid to an acquiring institution was considered nontaxable. In addition, in some cases, acquiring institutions could carry over certain losses and tax attributes of the acquired troubled institutions to reduce their own tax liability. The effect of these special tax benefits was to reduce the amount of FSLIC assistance payments required by an acquiring institution for a given transaction because of the value of tax benefits associated with the transaction. Thus, total assistance received by an acquiring institution consisted of both FSLIC payments and the value of these tax benefits. Because these tax benefits represented a reduction in general Treasury receipts rather than direct costs to FSLIC, we are presenting tax benefits as indirect costs associated with FSLIC’s assistance transactions. Of the $7.5 billion in estimated tax benefits, $3.1 billion has been realized through December 31, 1995. The remaining $4.4 billion represents an estimate of the future tax benefits that could be realized by acquiring institutions in the future. However, the amount of future tax benefits depends greatly upon the future actions and profitability of the acquirers. For example, reduced or enhanced earnings, institutional acquisitions, and changes in corporate control would all affect acquirers’ taxable income or the amount of tax benefits allowed to offset such taxable income in the future. The current estimate of future tax benefits is based on assumptions which are currently deemed most likely to occur in the future. However, if conditions change, the amount of future estimated tax benefits realized could be substantially higher or lower than the estimated $4.4 billion. Remaining Fiscal Implications of the Savings and Loan Crisis Although most of the direct and indirect costs of the savings and loan crisis had been funded or provided for through December 31, 1995, significant fiscal implications remain as a result of the crisis. Substantial funds were borrowed through bonds specifically designed to provide funding for a portion of the direct costs. Both taxpayers and the industry are paying financing costs on those bonds. In addition, a significant portion of direct costs were paid from appropriations at a time when the federal government was operating with a sizable budget deficit. Therefore, it is arguable that additional borrowing was incurred. In view of these circumstances, we are presenting information on the known and estimated interest expense associated with financing the crisis because the future stream of payments associated with interest will have continuing fiscal implications for taxpayers and the savings and loan industry. An additional fiscal implication is that SAIF is currently undercapitalized and the savings and loan industry continues to pay high insurance premiums to build the fund. FICO and REFCORP Bonds In 1987, the Congress established FICO, which had the sole purpose of borrowing funds to provide financing to FSLIC. FICO provided funding for FSLIC-related costs by issuing $8.2 billion of noncallable, 30-year bonds to the public. In 1989, the Congress established REFCORP to borrow funds and provide funding to RTC. REFCORP provided funding to the RTC for resolution losses by issuing $30.0 billion of noncallable, 30- and 40-year bonds to the public. The annual interest expense on the $38.2 billion of bonds issued by FICO and REFCORP has and will continue to have a significant impact on taxpayers and the savings and loan industry. The annual FICO bond interest is funded from the industry’s insurance premiums and represents an increasing burden on the savings and loan industry. In addition, the government’s portion of annual interest expense on the REFCORP bonds will continue to require the use of increasingly scarce budgetary resources. Annual interest on the FICO bonds is $793 million and is currently being paid from industry assessments and interest earnings on FICO’s cash balances. The annual interest obligation on the FICO bonds will continue through the maturity of the bonds in the years 2017 through 2019. The total nominal amount of interest expense over the life of the FICO bonds will be $23.8 billion. Annual interest expense on the REFCORP bonds is $2.6 billion. The Federal Home Loan Banks contribute $300 million annually to the payment of REFCORP interest expense, and the remaining $2.3 billion of annual interest expense is paid through appropriations. Annual interest expense will continue through the maturity of the REFCORP bonds in the years 2019, 2020, 2021, and 2030. The total nominal amount of interest expense over the life of the REFCORP bonds will be $88 billion. Estimated Treasury Interest Expense Associated With the Crisis The largest source of funding to pay the direct costs of the savings and loan crisis was provided by taxpayers as a result of legislation enacted to specifically deal with the crisis. This legislation was enacted during a period in which the federal government was financing—via deficit spending—a sizable portion of its regular, ongoing program activities and operations. Under these circumstances, it is arguable that substantial, incremental Treasury borrowing occurred in order to finance the taxpayer portion of the crisis. To arrive at an amount for estimated future interest associated with appropriations, we made various simplifying assumptions. For purposes of estimating Treasury interest expense associated with resolving the savings and loan crisis, we assumed that the entire amount of appropriations used to pay direct costs was borrowed. Various other simplifying assumptions were made regarding interest rates and the financing period. We assumed that the $99.3 billion in appropriations for the FSLIC Resolution Fund and the RTC would be financed for 30 years at 7 percent interest,with no future refinancing. Under these assumptions, approximately $209 billion in estimated interest payments would be needed over 30 years to cover the interest expense related to appropriations used to cover the direct costs of the crisis. Table 4 presents the known and estimated interest expense components associated with the financing mechanisms used to provide funds for the direct costs of the savings and loan crisis. Significant resources will be needed in the future to pay the known annual interest expense on the FICO and REFCORP bonds as well as the estimated Treasury interest expense related to the crisis. As shown in table 5, $20.4 billion, or 18 percent of the total nominal interest expense on FICO and REFCORP bonds has been paid through December 31, 1995. The remaining $91.4 billion, or 82 percent, will be funded in the future. Future interest expense of approximately $18 billion remains to be paid to cover the FICO bond interest. Currently, insurance premiums paid by certain SAIF-insured institutions are used to pay annual FICO bond interest expense. In 1995, the FICO interest expense represented about 69 percent of insurance premiums earned on SAIF’s FICO-assessable base. In recent years, the FICO-assessable base has been shrinking, thereby increasing the burden of the FICO interest expense relative to the size of the assessment base, and calling into question the future ability of the FICO-assessable base to cover the annual FICO interest expense. Future interest expense of approximately $73.4 billion remains to be paid on the REFCORP bonds. The Federal Home Loan Banks will continue to be responsible for paying $300 million each year toward the cost of REFCORP interest expense until the bonds mature. The remaining portion of the REFCORP bond interest expense will be paid with Treasury funds until the bonds mature in the years 2019, 2020, 2021, and 2030. For purposes of analyzing the timing of estimated Treasury interest expense on funds provided to pay the direct costs, we estimated that approximately $176 billion of the $209 billion in estimated Treasury interest expense, shown in table 5, related to future periods. Under these assumptions, future estimated Treasury interest would represent a significant claim on future federal budgetary resources. FIRREA created SAIF to insure deposits previously insured by the FSLIC, and set a designated reserve requirement of 1.25 percent of insured deposits. We consider the need to capitalize SAIF a remaining fiscal implication of the crisis because insurance premiums that could have been used to capitalize SAIF were used to pay a portion of the direct costs of the crisis,as well as annual interest expense on the FICO bonds. As a result, SAIF’s capitalization has been delayed, creating ongoing implications in terms of high deposit insurance premiums. In order to be fully capitalized, SAIF would have needed $8.9 billion in reserves based on the level of insured deposits at December 31, 1995. However, at that date, SAIF had reserves of only $3.4 billion, $5.5 billion below the designated reserve amount of $8.9 billion. Objectives, Scope, and Methodology preparing annual financial statements in conformity with generally establishing, maintaining, and assessing the internal control structure to provide reasonable assurance that the broad control objectives of FMFIA are met; and complying with applicable laws and regulations. We are responsible for obtaining reasonable assurance about whether (1) the financial statements are free of material misstatement and presented fairly, in all material respects, in conformity with generally accepted accounting principles and (2) RTC management’s assertion about the effectiveness of internal controls is fairly stated in all material respects and is based upon the criteria established under FMFIA. We are also responsible for testing compliance with selected provisions of laws and regulations and for performing limited procedures with respect to certain other information appearing in the financial statements. In order to fulfill these responsibilities, we examined, on a test basis, evidence supporting the amounts and disclosures in the financial statements; assessed the accounting principles used and significant estimates made by evaluated the overall presentation of the financial statements; obtained an understanding of the internal control structure related to safeguarding assets, compliance with laws and regulations, including the execution of transactions in accordance with management authority and financial reporting; tested relevant internal controls over safeguarding, compliance, and financial reporting and evaluated management’s assertion about the effectiveness of internal controls; and tested compliance with selected provisions of the following laws and regulations: section 21A of the Federal Home Loan Bank Act (12 U.S.C. 1441a) and Chief Financial Officers Act of 1990, sections 305 and 306 (Public Law 101-576). We did not evaluate all internal controls relevant to operating objectives as broadly defined by FMFIA, such as those controls relevant to preparing statistical reports and ensuring efficient operations. We limited our internal control testing to those controls necessary to achieve the objectives outlined in our opinion on RTC management’s assertion about the effectiveness of internal controls. Because of inherent limitations in any internal control structure, losses, noncompliance, or misstatements may nevertheless occur and not be detected. We also caution that projecting our evaluation to future periods is subject to the risk that controls may become inadequate because of changes in conditions or that the degree of compliance with controls may deteriorate. With the termination of RTC on December 31, 1995, an important phase of the savings and loan crisis ended. To provide an historical perspective on RTC and its role in resolving the crisis, we obtained and reviewed background information and data from RTC and FDIC. In addition, we obtained and analyzed audited financial information from the following entities which had varying roles in resolving the savings and loan crisis: FSLIC, FICO, RTC, REFCORP, FSLIC Resolution Fund, and SAIF. We conducted our audit from July 7, 1995, through June 7, 1996, in accordance with generally accepted government auditing standards. FDIC Comments and Our Evaluation FDIC provided written comments on a draft of this report because of its responsibility for RTC’s remaining assets and liabilities and its role in preparing RTC’s final financial statements. In FDIC’s comments, provided in appendix III, the Corporation’s Chief Financial Officer acknowledges the weaknesses in general controls over RTC’s computerized information systems and discusses the status of RTC and FDIC actions to correct them. We plan to evaluate the adequacy and effectiveness of those corrective actions as part of our audit of FDIC’s 1996 financial statements. The Chief Financial Officer’s comments also discuss FDIC’s involvement in RTC’s transition and FDIC’s plans in assuming responsibility for closing out RTC’s active and completed contracts. RTC’s Financial Statements Accounting and Information Management Division, Washington, D.C. Atlanta Regional Office Dallas Regional Office Denver Regional Office Kansas City Regional Office Related GAO Products Resolution Trust Corporation: Implementation of the Management Reforms in the RTC Completion Act (GAO/GGD-95-67, March 9, 1995) Resolution Trust Corporation: Evaluations Needed to Identify the Most Effective Land Sales Methods (GAO/GGD-95-43, April 13, 1995) 1993 Thrift Resolutions: RTC’s Resolution Process Generally Adequate to Determine Least Costly Resolutions (GAO/GGD-95-119, May 15, 1996) Resolution Trust Corporation: Management Improvements Reduce Risk But Transition Challenges Remain (GAO/T-GGD-95-163, May 16, 1995) Resolution Trust Corporation: Management Improvements Reduce Risk But Transition Challenges Remain (GAO/T-GGD-95-188, June 20, 1995) Inspectors General: Mandated Studies to Review Costly Bank and Thrift Failures (GAO/GGD-95-126, July 31, 1995) Resolution Trust Corporation: Performing Assets Sold to Acquirers of Minority Thrifts (GAO/GGD-96-44, December 22, 1995) The first copy of each GAO report and testimony is free. Additional copies are $2 each. Orders should be sent to the following address, accompanied by a check or money order made out to the Superintendent of Documents, when necessary. VISA and MasterCard credit cards are accepted, also. Orders for 100 or more copies to be mailed to a single address are discounted 25 percent. U.S. General Accounting Office P.O. Box 6015 Gaithersburg, MD 20884-6015 Room 1100 700 4th St. NW (corner of 4th and G Sts. NW) U.S. General Accounting Office Washington, DC Orders may also be placed by calling (202) 512-6000 or by using fax number (301) 258-4066, or TDD (301) 413-0006. Each day, GAO issues a list of newly available reports and testimony. To receive facsimile copies of the daily list or any list from the past 30 days, please call (202) 512-6000 using a touchtone phone. A recorded menu will provide information on how to obtain these lists. | Summary: Pursuant to a legislative requirement, GAO audited the Resolution Trust Corporation's (RTC) financial statements for the years ended December 31, 1995 and 1994. GAO also reviewed: (1) RTC internal control weaknesses; (2) RTC mission and its completion; (3) RTC costs and funding; and (4) the cost of resolving the savings and loan crisis. GAO found that: (1) RTC financial statements were reliable in all material aspects; (2) although RTC internal controls need improvement, they were effective in safeguarding assets, ensured that transactions were in accordance with management authority and material laws and regulations, and ensured that there were no material misstatements; and (3) there was no material noncompliance with applicable laws and regulations. GAO also found that: (1) RTC essentially accomplished its mission of closing insolvent institutions, liquidating institution assets, insuring depositor accounts, and bringing many thrift violators to justice; (2) the estimated cost of RTC activities totalled $87.9 billion; (3) RTC contractor control weaknesses and performance problems could adversely affect receivership recoveries; (4) all of the $160.1 billion in estimated direct and indirect costs of RTC and Federal Savings and Loan Insurance Corporation activities have been provided for as of December 31, 1995; (5) the cost of present and future litigation resulting from the savings and loan crisis is unknown; (6) the annual interest expense on the $38.2 billion in Financing Corporation and Resolution Funding Corporation bonds will continue to significantly impact taxpayers and the savings and loan industry; and (7) because the Savings Association Insurance Fund has not been fully capitalized, thus deposit insurance premiums have remained high. | 7,674 | 401 | gov_report | en |
Summarize: THE TECHNICAL FIELD OF THE INVENTION [0001] The invention relates to the design of medication delivery systems with a view to compactness, reliability, weight and production cost. [0002] The invention relates specifically to: A portable medication delivery device comprising a medication cartridge having an outlet and a movable piston, and a housing for holding said cartridge, and a flexible piston rod being operable to engage and displace said piston along an axis of said cartridge, and guiding means for bending said piston rod away from said axis, and actuating means, and driving means for transferring movement from said actuating means to said piston rod, said driving means including a driving wheel for displacing the piston rod, said flexible piston rod comprising regularly spaced first members adapted to mechanically cooperate with corresponding second members on said driving wheel. DESCRIPTION OF RELATED ART [0003] The following account of the prior art relates to one of the areas of application of the present invention, medication delivery systems for self-treatment of a disease. [0004] In a medication delivery system for self-treatment of a disease such as diabetes, safety and convenience in the handling of the injection or infusion are of major importance. One very important aspect of this is compactness of the device. Another very important aspect for a user is to ensure that a correct intended dose is delivered. For the manufacturer of the devices aspects such as production control (testability) and economical solutions are important. A handy, i.e. small volume and lightweight, device that is highly reliable and at the same time economical in production is in demand. [0005] U.S. Pat. No. 5,637,095 discloses a medication infusion pump with a flexible drive plunger comprising a spring tape. In one preferred form of the invention, the spring tape is wrapped or coiled onto a take-up spool within the pump housing. Drive means such as a drive capstan roller and associated pinch roller engage and advance the spring tape under control of the pump drive motor to correspondingly engage and advance the piston to deliver medication to the patient in a programmed manner. [0006] EP-A-0 110 687 discloses a portable fluid infusion device including a flexible piston rod assembly. Attached to the piston is a flexible pusher tape, reversely bent in a U-shape at the upstream or open end of the syringe so that the distal end of the tape is adjacent to and in a position to ride upon the cylindrical body of the syringe as the piston is moved within the body of the syringe. The flexible pusher tape is made of a readily flexible plastics material such as a molded polypropylene. A series of parallel grooves are preformed in the face of the plastics tape along the narrow center portion thereof. The outwardly extending face of the tape is engaged by the helical drive screw at the part of the piston rod, which is deflected away from the axis of the cartridge. The rotation of the screw by a DC motor propels the tape in its U-shaped path to drive the piston. DISCLOSURE OF THE INVENTION [0007] The problems of the prior art are that the driving means are located relatively far from the piston and require a relatively thick piston rod having a relatively high friction with the housing and the cartridge, thus requiring a relatively high power consuming motor to advance it and possibly introducing inaccuracies in the displacement of the piston, or that the means for advancing the tape-shaped piston rod, the take-up spool storing the unused part of the tape, and the drum or wheel that supports the U-shaped path of the tape are individual self-contained functional units, yielding a relatively complex solution. [0008] The object of the present invention is to provide a medication delivery system that combines compactness with an improved accuracy. [0009] This is achieved according to the invention in that said flexible piston rod exhibits a linear or approximately linear path between said driving wheel and said piston. [0010] In the present context, the term ‘medication delivery system’ is taken to mean an injector type device (such as a pen injector or a jet injector) for delivering a discrete dose of a liquid medication (possibly in the form of small drops) or a medication pump for continuous delivery of a liquid medication—in both cases optionally in combination with relevant electronic monitoring and control and possibly communications units. [0011] In the present context the term ‘piston’ is taken to mean a displaceable plate or cylinder that fits tightly against the inner walls of a cartridge. A surface of the piston that faces the inner part of the cartridge and which may be brought into contact with the contents of the cartridge is termed ‘the inner surface of the piston’, and the opposite side of the piston is termed ‘the outer surface of the piston’. In cooperation with a ‘piston rod’ that is engaged with ‘the outer surface of the piston’, the ‘piston’ may be displaced and used to apply pressure to a surface of the contents of the cartridge being in contact with ‘the inner surface of the piston’, thus e.g. delivering a dose through the outlet of the cartridge, if the piston is displaced in the direction towards the outlet. In the present context, the term ‘piston’ may also apply to a movable wall or membrane that engages with a plunger being an integral part of the piston rod. [0012] In the present context, the term ‘piston rod being operable to engage and displace said piston’is taken to mean that the piston rod may or may not be fixed to the movable wall, but that in both cases it has the ability to displace the piston at least in a direction towards the outlet of the cartridge. [0013] In the present context, the term ‘driving wheel’ is taken to mean the part of the driving means that cooperates with the activating means (e.g. an electromotor) and the piston rod to transfer the movement of the activating means to a displacement of the piston rod. It may take the form of a gear wheel or drum or any other appropriate form that may be adapted to cooperate with the piston rod. A gear box may be inserted between the activating means and the driving wheel. [0014] An advantage of having a linear path of the piston rod between the driving wheel and the piston is that the risk of introducing errors in the displacement of the piston by the rod is reduced because a more direct drive of the piston is provided (i.e. the sources of mechanical inaccuracies from a remotely located driving wheel due to the curved path, extra friction of the guiding means, etc. are eliminated). Related advantages of this are that an improved dose accuracy may be achieved and that the requirements with respect to the power of the activating means are reduced, leading to a possible reduction in weight and volume of the device. [0015] A further improved dose accuracy may be achieved by combining the present invention with the invention disclosed in our co-pending patent application A medication delivery system with improved dose accuracy incorporated herein by reference. [0016] Other advantages of the invention are that it uses a simple principle for transferring rotational movement of the actuating means to translatory movement of the piston rod, a principle that may be implemented in many different ways depending on the design constraints as regards space and materials. It allows the construction of a relatively compact medication device (utilizing e.g. a 180 degrees curve of the path of the piston rod), using a relatively thin piston rod yielding the benefits of a low weight and a potentially economical solution that is suited for large-scale production. [0017] When said flexible piston rod remains in a fully reversible elastic state during cooperation with said driving wheel and said guiding means, it is ensured that the piston rod is able to adjust to the various curvatures of the driving and guiding means and cooperate therewith, without being irreversibly deformed. [0018] When the surface of said flexible piston rod in a transversal cross-section is adapted to follow the surface of said driving wheel fully or partially, it is ensured that the change of the cross-section of the piston rod during its bending cooperation with the driving and guiding means is accounted for to ensure a seamless cooperation between the rod and the driving and guiding means. [0019] When said guiding means also work as a take-up spool for storing the unused part of the piston rod, it is ensured that an especially compact and economical solution is provided. [0020] When said driving wheel and said guiding means are the same physical unit, it is ensured that an especially simple, compact and economical solution is provided. [0021] When said driving wheel is located after said guiding means in a downstream direction towards said cartridge, it is ensured that the driving means are located as close as possible to the piston to be displaced, thus minimizing the sources of errors and improving dose accuracy. [0022] In the present context, the term ‘downstream’ is taken to mean in a direction of the movement of the piston when medication is expelled from the cartridge, said direction also defining a ‘longitudinal direction’ of the piston rod. [0023] When said piston rod touches the inner walls in an axial direction of said cartridge at one or more points in a transversal cross-section of said piston rod, it is ensured that the ability of the rod to withstand an axial pressure is improved. [0024] In the present context ‘the inner walls of the cartridge’ are taken to mean the interior surfaces of the walls of the cartridge being in contact with the medication. ‘A transversal cross-section of the piston rod’ is taken to mean a cross-section of the rod that is perpendicular to the longitudinal direction of the piston rod, i.e. for the part of the rod between the driving wheel and the piston, a direction perpendicular to the direction defined thereby (i.e. perpendicular to the axial direction of the cartridge). [0025] When the longitudinal edges of said piston rod in the cartridge touch the inner walls of said cartridge, it is ensured that the piston rod is guided when displacing the piston in the cartridge, improving the ability to withstand an axial pressure. [0026] In the present context the term ‘the longitudinal edges of said piston rod in the cartridge’ refers to the edges of the rod in a direction defined by the longitudinal direction of the piston rod. [0027] When said flexible piston rod is tape-shaped, it is ensured that a solution that is convenient from a production point of view and which is well suited for coiling is provided, and that an improved flexibility in the physical design of the medication delivery system is introduced. [0028] In the present context the term ‘tape-shaped’in connection with ‘piston rod’ is taken to mean that the cross-section of the rod perpendicular to its longitudinal direction is ‘wider’ than its ‘height’. It does not have to take the form of a rectangular cross-section but could be grooved or toothed or wave-shaped or convex or concave or something else that might be convenient from a design point of view. [0029] When said flexible piston rod consists of two separate tape-shaped bodies that are joined together at one or more points in a transversal cross-section, it is ensured that a greater axial pressure may be applied to the rod compared to a ‘single layer’ solution, in other words that thinner materials may be used, resulting in lower weight and reduced costs and thus a greater degree of freedom in the design of the medication delivery device. Further, the piston rod may be subject to a sharper bend than a corresponding one-layer rod. A tape-shaped piston rod that is sufficiently stiff in itself (i.e. without touching the inner walls of the cartridge) may thus be provided. [0030] In a preferred embodiment said two separate tape-shaped bodies are joined together in the central point of a transversal cross-section. [0031] In a preferred embodiment the distance between said two tape-shaped bodies increases with increasing distance from said central point when viewed in a transversal cross-section in a relaxed state. [0032] In the present context the term ‘in a relaxed state’ refers to a situation in which no external forces (other than gravity) are applied to the rod. [0033] When said two separate tape-shaped bodies are joined together at the edges of a transversal cross-section, it is ensured that an economical and light-weight solution that is well suited for production in larger quantities is provided, and which may be bent around a relatively smaller minimum radius of curvature. [0034] In a preferred embodiment, said edges are joined by applying a coating layer to the surface of said separate tape-shaped bodies. This has the advantage (in addition to joining the two bodies by forming a continuous and flexible hinge between their corresponding longitudinal edges) of yielding a very even surface of the rod and provides the possibility of applying a coating that is especially appropriate for the application in question, e.g. that it minimizes friction, that it improves corrosion resistance, etc. [0035] In a preferred embodiment said two tape-shaped bodies describe an eye-shaped path when viewed in a transversal cross-section in a relaxed state. [0036] In a preferred embodiment said first members on said flexible piston rod comprise protruding members and said corresponding second members on said driving wheel comprise receiving members. In an embodiment thereof, the driving wheel comprises e.g. regularly spaced indentations that interact with corresponding protrusions on the piston rod to displace the piston rod. This has the advantage that the protrusions are ‘hidden’ in the driving wheel when the piston rod engages the wheel, possibly reducing the free space needed around the driving wheel (thus minimizing the volume of the construction). [0037] In a preferred embodiment said first members comprise receiving members and said corresponding second members comprise protruding members. [0038] In a preferred embodiment said first members comprise individually isolated indentations and said corresponding second members comprise individually isolated projecting members. [0039] When said first members comprise individually isolated through holes and said corresponding second members comprise individually isolated projecting members, it is ensured that a secure grip between piston rod and driving wheel is provided. [0040] In a preferred embodiment said regularly spaced members are located along a centerline in the longitudinal direction of said flexible piston rod. [0041] When said first members comprise individually isolated cuts located at least along one periphery in the longitudinal direction of said flexible piston rod and said corresponding second members comprise individually isolated projecting members, it is ensured that a secure grip between piston rod and driving wheel is provided. Further, a greater axial stiffness of the rod is achieved. [0042] When the piston rod is at least partially made of a plastics material, it is ensured that a solution combining the benefits of using a plastics material (e.g. corrosion resistance) with those of other materials (e.g. greater mechanical stability, stiffness, etc.) may be provided. [0043] In a preferred embodiment the piston rod is at least partially made of a metallic material. BRIEF DESCRIPTION OF THE DRAWINGS [0044] The invention will be explained more fully below in connection with a preferred embodiment and with reference to the drawings, in which: [0045] [0045]FIG. 1 shows a medication delivery system according to the invention where the driving wheel is located next to the medication cartridge, [0046] [0046]FIGS. 2. a - 2. b show an embodiment of the invention where driving wheel and guiding means are one physical unit, [0047] [0047]FIGS. 3. a - 3. d show various embodiments of a tape-shaped piston rod according to the invention, [0048] [0048]FIGS. 4. a - 4. c show a tape-shaped piston rod and corresponding driving wheel according to the invention for which a coiling of the tape on the driving wheel is possible, [0049] [0049]FIG. 5 shows a medication delivery system according to the invention where the driving wheel in the form of a screw is located between the guiding means and the medication cartridge, [0050] [0050]FIGS. 6. a - 6. b show some possible designs of a piston rod according to the invention, and [0051] [0051]FIGS. 7. a - 7. b show some possible designs of a piston rod and a driving drum according to the invention. [0052] The figures are schematic and simplified for clarity, and they just show details which are essential to the understanding of the invention, while other details are left out. Throughout, the same reference numerals are used for identical or corresponding parts. DETAILED DESCRIPTION OF EMBODIMENTS [0053] [0053]FIG. 1 shows a medication delivery system according to the invention where the driving wheel is located next to the medication cartridge. [0054] In the embodiment of the medication delivery system 1 in FIG. 1, a medication cartridge 11 (possibly a replaceable one) with a piston 112 and an outlet 111 to which a needle 22 (possibly replaceable) may be fixed, is shown to cooperate with a piston rod 13 in the form of a tape, the tape being arcuate (cf. correspondingly FIG. 2. b ) in a transversal cross-section. The cartridge 11 is removably fixed to a cartridge holder 12. The piston rod 13 may be displaced along a longitudinal axis 113 of the cartridge 11. A downstream direction is defined by the arrow 1131. The movement of the piston rod 13 is activated by an electromotor 15 whose rotational movement is transferred to a linear displacement of the piston rod by suitable driving means, the driving means comprising inter alia a driving wheel 17 with regularly spaced protruding members 171 that are adapted to cooperate with corresponding regularly spaced openings (cf. 331 in FIGS. 3. c and 3. d ) on the piston rod. The piston rod 13 is bent to make a 180 degrees U-turn over a first guiding wheel 14. A second guiding wheel 18 ensures a proper contact between the piston rod and the driving wheel 17. In the embodiment of FIG. 1, the faces of the second guiding wheel 18 and the driving wheel 17 that receive the tape-shaped piston rod 13 have a concave face and a corresponding convex face, respectively. This has the effect that the piston rod acquires its normal arcuate form in a transversal direction of the rod appropriately adjusted to the diameter of the medication cartridge (e.g. so that the edges of the piston rod (and not the central part of the rod) touch the inner walls of the cartridge (cf. FIG. 2. b ). The receiving faces of the guiding wheels 14, 18 and driving wheel 17 may of course take other forms that are convenient from a design point of view. [0055] In an embodiment of the invention, the piston rod 13 is adapted to elastically adjust fully or partially to the shape of the surfaces of the driving wheel 17 and the guiding wheel 14 along its path of contact with said wheels. Alternatively, the surfaces of the wheels are adapted to match fully or partially the shape of the side of the piston rod that engages with the wheels when the piston rod is bent around the wheel in question. This applies to the longitudinal as well as the transversal directions of the rod. In the longitudinal direction, the smallest diameter of the wheel is limited by the smallest diameter around which the rod may be elastically bent (i.e. reversibly). In the transversal direction, for the embodiment in FIG. 6. a, for example, this means that the eye-shaped cross-sectional view ‘collapse’ to a shape that follows the wheel in question. [0056] In another embodiment of the invention, the width of the driving wheel (and possible guiding wheels) is smaller than the width of the tape-shaped rod when viewed in a transversal cross-section (cf. FIG. 7. a ). In this case, a piston rod with centrally located (cf. e.g. FIG. 3. a, 3. c or 6. a ) or nearly centrally located (cf. FIG. 3. d or 6. b ) is used. [0057] In an embodiment of the invention said first guiding wheel 14 is substituted by a fixed guideway (providing an equivalent bending of the piston rod), optionally coated with a layer that ensures a low friction between guideway and piston rod. The transversal cross-section of the guideway is adapted to match fully or partially the shape of the side of the piston rod that engages with the guideway when the piston rod is bent around it during its normal operation. [0058] When the medication cartridge is replaceable, it is ensured that the major part of the medication device may be used again and again only by inserting a new cartridge (and possibly a new needle in the case of an injection device) when the contents of the medication cartridge has been ejected or when another medication is to be used, i.e. e.g. in the situation of a person's self-treatment of a disease (e.g. diabetes) that requires frequent delivery of medication (e.g. insulin) over an extended period of time. If the replaceable cartridge contains a fully functioning piston (and possibly a corresponding piston rod), a convenient and flexible solution is provided, where the medication cartridge may be replaced in a quick and hygienically safe way. [0059] To ensure that the piston rod follows the guiding wheel 14 along the relevant part thereof, the ‘upstream’ end of the piston rod (i.e. the end of the rod that does not engage the piston) may be connected to a part of the medication device that is held fixed relative to the cartridge by a salient element (e.g. a spring, not shown) whose one end is tied to the piston rod and whose other end is held fixed. [0060] Typically, the medication delivery process including analysis and use of historical data concerning the device and user in question, diagnostic proposals, error correction, etc. is governed by appropriate processing and communications units. [0061] In the embodiment in FIG. 1. a the driving means include appropriate means 16 for transferring movement from the electromotor 15 to the driving wheel 17, e.g. in the form of a gear box. The electromotor is controlled by a processing unit 19 (including relevant memory means). The processing unit 19 may exchange information with the user and other systems via I/O-means 20 (comprising e.g. a display, keypad, and relevant communications interfaces). The electronic units are powered from the energy source 21 (e.g. a battery pack or an interface to external energizing means). [0062] A housing 10 for protecting and optionally supporting the piston rod at its 180 degrees path and for covering the electromotor and other vital parts of the device is provided. [0063] The housing 10, the cartridge holder 12, the means 16 for transferring movement from the electromotor to the driving wheel, the electromotor 15, the processing unit 19, the I/O-means 20 and the energy source 21 are not included in FIGS. 2 and 5, but are implied. [0064] [0064]FIGS. 2. a - 2. b show an embodiment of the invention where driving wheel and guiding means are one physical unit. [0065] In FIG. 2. a only the central features of the invention are illustrated. FIG. 2. a shows a driving wheel 17 with regularly spaced teeth 171 that cooperate with corresponding indentations on the piston rod 13. When the driving wheel 17 is activated in a counterclockwise direction by the actuating means (not shown), the piston rod 13 is displaced in a downstream direction and acts to displace the piston 112 to expel medication from the cartridge 11 through the outlet 111 and an attached needle (not shown). By allowing the driving wheel to create the 180 degrees bending of the piston rod, a particularly simple embodiment is provided. Additionally, this embodiment is more compact and lighter. [0066] [0066]FIG. 2. b shows a cross-sectional view of the medication cartridge 11 and the piston rod 13 along the line BB. The curved cross-section of the piston rod 13 is apparent. The edges 131, 132 of the tape-shaped piston rod glide on the inner walls of the cartridge 11, yielding a mechanically more stable construction in that a greater force may be applied to the tape rod, without it bending out of the linear path from the edge of the driving wheel to the point of engagement of the piston rod with the piston. [0067] [0067]FIGS. 3. a - 3. d show various embodiments of a tape-shaped piston rod according to the invention. [0068] [0068]FIG. 3. a shows an embodiment of a tape-shaped piston rod 33 with parallel grooves 330 in a transversal (or nearly transversal) direction of the tape-shaped piston rod in a central part on the side of the tape that cooperates with a corresponding driving screw (cf. 17 in FIG. 5). The grooves may optionally extend through the material to form slots for cooperation with corresponding threads on the driving wheel. [0069] [0069]FIG. 3. b shows an embodiment of a tape-shaped piston rod 33 with regularly spaced cut-outs 332 in each of the longitudinal edges of the tape for cooperation with corresponding projecting pins on the driving wheel (not illustrated). Put differently, the tape-shaped piston rod is provided with regularly spaced projecting members along its longitudinal edges and the driving wheel has corresponding receiving members to provide a secure grip of the piston rod when the rod engages with the driving wheel. This embodiment yields a very good grip between tape and wheel and a very accurate positioning in a transversal direction of the tape, both contributing to a more accurate dosage. It further provides a piston rod that is able to withstand a larger axial pressure (without bending) than corresponding solutions depicted in FIGS. 3. c and 3. d. [0070] [0070]FIG. 3. c shows an embodiment of a tape-shaped piston rod 33 with regularly spaced, centrally located rectangular through holes 331 in the tape for cooperation with corresponding projecting pins on the driving wheel (e.g. 171 in FIGS. 1 and 2). [0071] [0071]FIG. 3. d shows an embodiment of a tape-shaped piston rod 33 with two rows of regularly spaced, circular through holes 331 located symmetrically around the longitudinal centre line of symmetry of the tape for cooperation with corresponding projecting pins on the driving wheel (not shown). [0072] [0072]FIGS. 4. a - 4. c show a tape-shaped piston rod and corresponding driving wheel according to the invention for which a coiling of the tape on the driving wheel is possible. [0073] The piston 33 comprises a tape with centrally situated, regularly spaced circular holes 331 adapted to cooperate with corresponding protruding circular cylindrical members 361 on the driving drum 36. The driving drum is activated by an electromotor (not shown) through appropriate driving means (e.g. a gear box, not shown). The holes are shown to be positioned along a centerline of the tape, but may of course be located at one or both longitudinal edges of the tape (cf. FIG. 3. b ) or along a line off the centre line or in any other convenient way as long as the protruding means on the driving drum follow a corresponding pattern. Likewise, the individual holes and corresponding protruding members may take on any convenient form, e.g. edged as opposed to circular, as long as the holes in the tape and the protruding members on the driving drum correspond. [0074] [0074]FIGS. 4. a and 4. b show orthogonal plane views of the piston rod and driving drum, whereas FIG. 4. c shows a perspective view of a coiled piston rod and illustrate the fact that the unused part of the tape/piston rod may be stored on the driving drum, yielding a simple and compact solution. [0075] [0075]FIG. 5 shows a medication delivery system according to the invention where the driving wheel in the form of a screw is located between the guiding means and the medication cartridge. [0076] [0076]FIG. 5 shows a driving screw 17 that cooperates with corresponding grooves (cf. 330 in FIG. 3. a ) on the piston rod 13. When the driving wheel 17 is activated by the actuating means (not shown), the piston rod 13 is displaced in a downstream direction and acts to displace the piston 112 to expel medication from the cartridge 11 through the outlet and an attached needle (not shown). The piston rod makes a 180 degrees bend over a guiding wheel 14. A second guiding wheel 18 ensures a proper contact between the piston rod and the driving screw 17. [0077] [0077]FIG. 6 shows some possible designs of a piston rod according to the invention. [0078] The piston rod may be made in many different forms according to design requirements (materials, stiffness, weight, corrosion, etc.) and cost. The rod may be made of one longitudinal piece of material or alternatively be composed of several layers joined together at one or more points in a transversal cross-section of the tape. In the longitudinal direction, the tape may be joined together in isolated (possibly regularly spaced) ‘points’ or continuously. The joining may be performed by welding or adhesive techniques or any other appropriate joining technique. [0079] [0079]FIG. 6. a shows a preferred embodiment of a tape-shaped piston rod 33, where the rod is made of two identical longitudinal pieces 335, 336 of foil that are joined together along their edges 131, 132 and have centrally located 334, regularly spaced openings 331 for cooperation with a driving wheel. The two individual pieces of tape have an arcuate cross-section in a relaxed state, thus forming an eye-shaped cross-section when joined at the edges. In a preferred embodiment the individual pieces of tape are made of a salient metallic material of 0.5 mm thickness and the joining is achieved by coating the outer surface with a ductile polymer layer. If appropriate, the layer may be applied to the inner surfaces of the rod or only along the joining lines of the rod. Alternatively, a ductile adhesive tape may be applied along the joining lines of the rod. [0080] In another preferred embodiment, as depicted in FIG. 6 b, the rod 33 is made of two identical longitudinal pieces 337, 338 of foil that are joined together along their centre lines 334 to give an X-shaped cross-section and having two rows of regularly spaced holes 3311, 3312 for cooperation with a driving wheel. The holes are located on each side of the central point of the tape when viewed in a transversal cross-section of the rod. The holes are shown to be circular and quadratic, respectively, but they may take any form that is appropriate for cooperation with a driving wheel. Likewise indentations may be used depending on material thickness as long as a sufficient grip with the driving wheel is ensured. [0081] [0081]FIGS. 7. a - 7. b show some possible designs of a piston rod and a driving drum according to the invention. [0082] [0082]FIG. 7. a shows an embodiment of the invention where the driving drum 17 with regularly spaced projecting pins 171 has a width 173 in the direction of its axis of symmetry that is much smaller than the width 335 of the tape-shaped piston rod 33. The piston rod 33 has regularly spaced openings 331 located along a centre line 334 adapted to cooperate with the projecting pins 171 of the driving drum 17. The piston rod has longitudinal edges 131, 132. The piston rod has an arcuate transversal cross-section in a relaxed state. The surface 172 of the driving drum facing the corresponding surface 133 of the piston rod (shown in FIG. 7. a in its relaxed state) is flat (linear as opposed to arcuate), but may take any convenient form as long as its width 172 is sufficiently small compared to the width 335 of the rod and the projecting pins provide a sufficient grip with the openings of the piston rod. [0083] [0083]FIG. 7. b shows an embodiment of the invention where the driving drum 17 with regularly spaced projecting pins 171 has a width 173 in the direction of its axis of symmetry that is comparable to the width 335 of the tape-shaped piston rod 33. Again, the piston rod 33 has regularly spaced openings 331 located along a centre line 334, the rod having longitudinal edges 131, 132. The openings 331 are adapted to cooperate with the projecting pins 171 of the driving drum 17. The piston rod has an arcuate transversal cross-section in a relaxed state. The surface 172 of the driving drum facing the corresponding surface 133 of the piston rod is arcuate and adapted to match the curvature of the piston rod fully or partially in a transversal cross-section, when the rod is brought into contact with and forced to follow a radial path of the drum (i.e. when the projecting pins of the drum cooperate with the openings of the piston rod). The arcuate surface 133 of the piston rod in a relaxed state is shown in FIG. 7. b. In general, the radius of curvature of the surface 172 of the driving drum contacting the piston rod is greater than the radius of curvature of the corresponding surface 133 of the transversal cross-section of the piston rod in a relaxed state, as indicated in FIG. 7. b. In a special embodiment the radius of curvature of the surface 172 of the driving drum contacting the piston rod is infinite (i.e. the surface is linear). [0084] The piston rod and driving and guiding members should be designed in such a way that the piston rod, when cooperating with the said members, remains in a fully reversible, elastic mode of deformation (in its longitudinal as well as its transversal cross-sections). This may be achieved by a proper choice of materials and geometrical dimensions. [0085] Some preferred embodiments have been shown in the foregoing, but it should be stressed that the invention is not limited to these, but may be embodied in other ways within the subject-matter defined in the following claims. | Summary: The invention relates to: A portable medication delivery device ( 1 ) comprising a medication cartridge ( 11 ) having an outlet ( 111 ) and a movable piston ( 112 ), and a housing ( 12 ) for holding said cartridge, and a flexible piston rod ( 13 ) being operable to engage and displace said piston along an axis ( 113 ) of said cartridge, and guiding means ( 14 ) for bending said piston rod away from said axis, and actuating means ( 15 ), and driving means ( 16, 17 ) for transferring movement from said actuating means to said piston rod, said driving means including a driving wheel ( 17 ) for displacing the piston rod ( 13 ), said flexible piston rod comprising regularly spaced first members ( 330; 331; 332 ) adapted to mechanically cooperate with corresponding second members ( 171 ) on said driving wheel. The object of the present invention is to provide a medication delivery system that combines compactness with an improved accuracy. The problem is solved in that said flexible piston rod ( 13 ) exhibits a linear or approximately linear path between said driving wheel ( 17 ) and said piston ( 112 ). This has the advantage of yielding a compact, low-weight device with an improved dose accuracy. The invention may e.g. be used in injection or infusion devices for a person's self-treatment of a disease such as diabetes. | 8,151 | 353 | big_patent | en |
Summarize: Two days are passed. It is a summer evening; the coachman has set me down at a place called Whitcross; he could take me no farther for the sum I had given, and I was not possessed of another shilling in the world. The coach is a mile off by this time; I am alone. At this moment I discover that I forgot to take my parcel out of the pocket of the coach, where I had placed it for safety; there it remains, there it must remain; and now, I am absolutely destitute. Whitcross is no town, nor even a hamlet; it is but a stone pillar set up where four roads meet: whitewashed, I suppose, to be more obvious at a distance and in darkness. Four arms spring from its summit: the nearest town to which these point is, according to the inscription, distant ten miles; the farthest, above twenty. From the well-known names of these towns I learn in what county I have lighted; a north-midland shire, dusk with moorland, ridged with mountain: this I see. There are great moors behind and on each hand of me; there are waves of mountains far beyond that deep valley at my feet. The population here must be thin, and I see no passengers on these roads: they stretch out east, west, north, and south--white, broad, lonely; they are all cut in the moor, and the heather grows deep and wild to their very verge. Yet a chance traveller might pass by; and I wish no eye to see me now: strangers would wonder what I am doing, lingering here at the sign-post, evidently objectless and lost. I might be questioned: I could give no answer but what would sound incredible and excite suspicion. Not a tie holds me to human society at this moment--not a charm or hope calls me where my fellow-creatures are--none that saw me would have a kind thought or a good wish for me. I have no relative but the universal mother, Nature: I will seek her breast and ask repose. I struck straight into the heath; I held on to a hollow I saw deeply furrowing the brown moorside; I waded knee-deep in its dark growth; I turned with its turnings, and finding a moss-blackened granite crag in a hidden angle, I sat down under it. High banks of moor were about me; the crag protected my head: the sky was over that. Some time passed before I felt tranquil even here: I had a vague dread that wild cattle might be near, or that some sportsman or poacher might discover me. If a gust of wind swept the waste, I looked up, fearing it was the rush of a bull; if a plover whistled, I imagined it a man. Finding my apprehensions unfounded, however, and calmed by the deep silence that reigned as evening declined at nightfall, I took confidence. As yet I had not thought; I had only listened, watched, dreaded; now I regained the faculty of reflection. What was I to do? Where to go? Oh, intolerable questions, when I could do nothing and go nowhere!--when a long way must yet be measured by my weary, trembling limbs before I could reach human habitation--when cold charity must be entreated before I could get a lodging: reluctant sympathy importuned, almost certain repulse incurred, before my tale could be listened to, or one of my wants relieved! I touched the heath: it was dry, and yet warm with the heat of the summer day. I looked at the sky; it was pure: a kindly star twinkled just above the chasm ridge. The dew fell, but with propitious softness; no breeze whispered. Nature seemed to me benign and good; I thought she loved me, outcast as I was; and I, who from man could anticipate only mistrust, rejection, insult, clung to her with filial fondness. To-night, at least, I would be her guest, as I was her child: my mother would lodge me without money and without price. I had one morsel of bread yet: the remnant of a roll I had bought in a town we passed through at noon with a stray penny--my last coin. I saw ripe bilberries gleaming here and there, like jet beads in the heath: I gathered a handful and ate them with the bread. My hunger, sharp before, was, if not satisfied, appeased by this hermit's meal. I said my evening prayers at its conclusion, and then chose my couch. {I said my evening prayers: p311.jpg} Beside the crag the heath was very deep: when I lay down my feet were buried in it; rising high on each side, it left only a narrow space for the night-air to invade. I folded my shawl double, and spread it over me for a coverlet; a low, mossy swell was my pillow. Thus lodged, I was not, at least--at the commencement of the night, cold. My rest might have been blissful enough, only a sad heart broke it. It plained of its gaping wounds, its inward bleeding, its riven chords. It trembled for Mr. Rochester and his doom; it bemoaned him with bitter pity; it demanded him with ceaseless longing; and, impotent as a bird with both wings broken, it still quivered its shattered pinions in vain attempts to seek him. Worn out with this torture of thought, I rose to my knees. Night was come, and her planets were risen: a safe, still night: too serene for the companionship of fear. We know that God is everywhere; but certainly we feel His presence most when His works are on the grandest scale spread before us; and it is in the unclouded night-sky, where His worlds wheel their silent course, that we read clearest His infinitude, His omnipotence, His omnipresence. I had risen to my knees to pray for Mr. Rochester. Looking up, I, with tear-dimmed eyes, saw the mighty Milky- way. Remembering what it was--what countless systems there swept space like a soft trace of light--I felt the might and strength of God. Sure was I of His efficiency to save what He had made: convinced I grew that neither earth should perish, nor one of the souls it treasured. I turned my prayer to thanksgiving: the Source of Life was also the Saviour of spirits. Mr. Rochester was safe; he was God's, and by God would he be guarded. I again nestled to the breast of the hill; and ere long in sleep forgot sorrow. But next day, Want came to me pale and bare. Long after the little birds had left their nests; long after bees had come in the sweet prime of day to gather the heath honey before the dew was dried--when the long morning shadows were curtailed, and the sun filled earth and sky--I got up, and I looked round me. What a still, hot, perfect day! What a golden desert this spreading moor! Everywhere sunshine. I wished I could live in it and on it. I saw a lizard run over the crag; I saw a bee busy among the sweet bilberries. I would fain at the moment have become bee or lizard, that I might have found fitting nutriment, permanent shelter here. But I was a human being, and had a human being's wants: I must not linger where there was nothing to supply them. I rose; I looked back at the bed I had left. Hopeless of the future, I wished but this--that my Maker had that night thought good to require my soul of me while I slept; and that this weary frame, absolved by death from further conflict with fate, had now but to decay quietly, and mingle in peace with the soil of this wilderness. Life, however, was yet in my possession, with all its requirements, and pains, and responsibilities. The burden must be carried; the want provided for; the suffering endured; the responsibility fulfilled. I set out. Whitcross regained, I followed a road which led from the sun, now fervent and high. By no other circumstance had I will to decide my choice. I walked a long time, and when I thought I had nearly done enough, and might conscientiously yield to the fatigue that almost overpowered me--might relax this forced action, and, sitting down on a stone I saw near, submit resistlessly to the apathy that clogged heart and limb--I heard a bell chime--a church bell. I turned in the direction of the sound, and there, amongst the romantic hills, whose changes and aspect I had ceased to note an hour ago, I saw a hamlet and a spire. All the valley at my right hand was full of pasture- fields, and cornfields, and wood; and a glittering stream ran zig-zag through the varied shades of green, the mellowing grain, the sombre woodland, the clear and sunny lea. Recalled by the rumbling of wheels to the road before me, I saw a heavily-laden waggon labouring up the hill, and not far beyond were two cows and their drover. Human life and human labour were near. I must struggle on: strive to live and bend to toil like the rest. About two o'clock p.m. I entered the village. At the bottom of its one street there was a little shop with some cakes of bread in the window. I coveted a cake of bread. With that refreshment I could perhaps regain a degree of energy: without it, it would be difficult to proceed. The wish to have some strength and some vigour returned to me as soon as I was amongst my fellow-beings. I felt it would be degrading to faint with hunger on the causeway of a hamlet. Had I nothing about me I could offer in exchange for one of these rolls? I considered. I had a small silk handkerchief tied round my throat; I had my gloves. I could hardly tell how men and women in extremities of destitution proceeded. I did not know whether either of these articles would be accepted: probably they would not; but I must try. I entered the shop: a woman was there. Seeing a respectably-dressed person, a lady as she supposed, she came forward with civility. How could she serve me? I was seized with shame: my tongue would not utter the request I had prepared. I dared not offer her the half-worn gloves, the creased handkerchief: besides, I felt it would be absurd. I only begged permission to sit down a moment, as I was tired. Disappointed in the expectation of a customer, she coolly acceded to my request. She pointed to a seat; I sank into it. I felt sorely urged to weep; but conscious how unseasonable such a manifestation would be, I restrained it. Soon I asked her "if there were any dressmaker or plain-workwoman in the village?" "Yes; two or three. Quite as many as there was employment for." I reflected. I was driven to the point now. I was brought face to face with Necessity. I stood in the position of one without a resource, without a friend, without a coin. I must do something. What? I must apply somewhere. Where? "Did she know of any place in the neighbourhood where a servant was wanted?" "Nay; she couldn't say." "What was the chief trade in this place? What did most of the people do?" "Some were farm labourers; a good deal worked at Mr. Oliver's needle-factory, and at the foundry." "Did Mr. Oliver employ women?" "Nay; it was men's work." "And what do the women do?" "I knawn't," was the answer. "Some does one thing, and some another. Poor folk mun get on as they can." She seemed to be tired of my questions: and, indeed, what claim had I to importune her? A neighbour or two came in; my chair was evidently wanted. I took leave. I passed up the street, looking as I went at all the houses to the right hand and to the left; but I could discover no pretext, nor see an inducement to enter any. I rambled round the hamlet, going sometimes to a little distance and returning again, for an hour or more. Much exhausted, and suffering greatly now for want of food, I turned aside into a lane and sat down under the hedge. Ere many minutes had elapsed, I was again on my feet, however, and again searching something--a resource, or at least an informant. A pretty little house stood at the top of the lane, with a garden before it, exquisitely neat and brilliantly blooming. I stopped at it. What business had I to approach the white door or touch the glittering knocker? In what way could it possibly be the interest of the inhabitants of that dwelling to serve me? Yet I drew near and knocked. A mild-looking, cleanly-attired young woman opened the door. In such a voice as might be expected from a hopeless heart and fainting frame--a voice wretchedly low and faltering--I asked if a servant was wanted here? "No," said she; "we do not keep a servant." "Can you tell me where I could get employment of any kind?" I continued. "I am a stranger, without acquaintance in this place. I want some work: no matter what." But it was not her business to think for me, or to seek a place for me: besides, in her eyes, how doubtful must have appeared my character, position, tale. She shook her head, she "was sorry she could give me no information," and the white door closed, quite gently and civilly: but it shut me out. If she had held it open a little longer, I believe I should have begged a piece of bread; for I was now brought low. I could not bear to return to the sordid village, where, besides, no prospect of aid was visible. I should have longed rather to deviate to a wood I saw not far off, which appeared in its thick shade to offer inviting shelter; but I was so sick, so weak, so gnawed with nature's cravings, instinct kept me roaming round abodes where there was a chance of food. Solitude would be no solitude--rest no rest--while the vulture, hunger, thus sank beak and talons in my side. I drew near houses; I left them, and came back again, and again I wandered away: always repelled by the consciousness of having no claim to ask--no right to expect interest in my isolated lot. Meantime, the afternoon advanced, while I thus wandered about like a lost and starving dog. In crossing a field, I saw the church spire before me: I hastened towards it. Near the churchyard, and in the middle of a garden, stood a well-built though small house, which I had no doubt was the parsonage. I remembered that strangers who arrive at a place where they have no friends, and who want employment, sometimes apply to the clergyman for introduction and aid. It is the clergyman's function to help--at least with advice--those who wished to help themselves. I seemed to have something like a right to seek counsel here. Renewing then my courage, and gathering my feeble remains of strength, I pushed on. I reached the house, and knocked at the kitchen-door. An old woman opened: I asked was this the parsonage? "Yes." "Was the clergyman in?" "No." "Would he be in soon?" "No, he was gone from home." "To a distance?" "Not so far--happen three mile. He had been called away by the sudden death of his father: he was at Marsh End now, and would very likely stay there a fortnight longer." "Was there any lady of the house?" "Nay, there was naught but her, and she was housekeeper;" and of her, reader, I could not bear to ask the relief for want of which I was sinking; I could not yet beg; and again I crawled away. Once more I took off my handkerchief--once more I thought of the cakes of bread in the little shop. Oh, for but a crust! for but one mouthful to allay the pang of famine! Instinctively I turned my face again to the village; I found the shop again, and I went in; and though others were there besides the woman I ventured the request--"Would she give me a roll for this handkerchief?" She looked at me with evident suspicion: "Nay, she never sold stuff i' that way." Almost desperate, I asked for half a cake; she again refused. "How could she tell where I had got the handkerchief?" she said. "Would she take my gloves?" "No! what could she do with them?" Reader, it is not pleasant to dwell on these details. Some say there is enjoyment in looking back to painful experience past; but at this day I can scarcely bear to review the times to which I allude: the moral degradation, blent with the physical suffering, form too distressing a recollection ever to be willingly dwelt on. I blamed none of those who repulsed me. I felt it was what was to be expected, and what could not be helped: an ordinary beggar is frequently an object of suspicion; a well-dressed beggar inevitably so. To be sure, what I begged was employment; but whose business was it to provide me with employment? Not, certainly, that of persons who saw me then for the first time, and who knew nothing about my character. And as to the woman who would not take my handkerchief in exchange for her bread, why, she was right, if the offer appeared to her sinister or the exchange unprofitable. Let me condense now. I am sick of the subject. A little before dark I passed a farm-house, at the open door of which the farmer was sitting, eating his supper of bread and cheese. I stopped and said-- "Will you give me a piece of bread? for I am very hungry." He cast on me a glance of surprise; but without answering, he cut a thick slice from his loaf, and gave it to me. I imagine he did not think I was a beggar, but only an eccentric sort of lady, who had taken a fancy to his brown loaf. As soon as I was out of sight of his house, I sat down and ate it. I could not hope to get a lodging under a roof, and sought it in the wood I have before alluded to. But my night was wretched, my rest broken: the ground was damp, the air cold: besides, intruders passed near me more than once, and I had again and again to change my quarters; no sense of safety or tranquillity befriended me. Towards morning it rained; the whole of the following day was wet. Do not ask me, reader, to give a minute account of that day; as before, I sought work; as before, I was repulsed; as before, I starved; but once did food pass my lips. At the door of a cottage I saw a little girl about to throw a mess of cold porridge into a pig trough. "Will you give me that?" I asked. {"Will you give me that?" I asked: p316.jpg} She stared at me. "Mother!" she exclaimed, "there is a woman wants me to give her these porridge." "Well lass," replied a voice within, "give it her if she's a beggar. T' pig doesn't want it." The girl emptied the stiffened mould into my hand, and I devoured it ravenously. As the wet twilight deepened, I stopped in a solitary bridle-path, which I had been pursuing an hour or more. "My strength is quite failing me," I said in a soliloquy. "I feel I cannot go much farther. Shall I be an outcast again this night? While the rain descends so, must I lay my head on the cold, drenched ground? I fear I cannot do otherwise: for who will receive me? But it will be very dreadful, with this feeling of hunger, faintness, chill, and this sense of desolation--this total prostration of hope. In all likelihood, though, I should die before morning. And why cannot I reconcile myself to the prospect of death? Why do I struggle to retain a valueless life? Because I know, or believe, Mr. Rochester is living: and then, to die of want and cold is a fate to which nature cannot submit passively. Oh, Providence! sustain me a little longer! Aid!--direct me!" My glazed eye wandered over the dim and misty landscape. I saw I had strayed far from the village: it was quite out of sight. The very cultivation surrounding it had disappeared. I had, by cross-ways and by- paths, once more drawn near the tract of moorland; and now, only a few fields, almost as wild and unproductive as the heath from which they were scarcely reclaimed, lay between me and the dusky hill. "Well, I would rather die yonder than in a street or on a frequented road," I reflected. "And far better that crows and ravens--if any ravens there be in these regions--should pick my flesh from my bones, than that they should be prisoned in a workhouse coffin and moulder in a pauper's grave." To the hill, then, I turned. I reached it. It remained now only to find a hollow where I could lie down, and feel at least hidden, if not secure. But all the surface of the waste looked level. It showed no variation but of tint: green, where rush and moss overgrew the marshes; black, where the dry soil bore only heath. Dark as it was getting, I could still see these changes, though but as mere alternations of light and shade; for colour had faded with the daylight. My eye still roved over the sullen swell and along the moor-edge, vanishing amidst the wildest scenery, when at one dim point, far in among the marshes and the ridges, a light sprang up. "That is an _ignis fatuus_," was my first thought; and I expected it would soon vanish. It burnt on, however, quite steadily, neither receding nor advancing. "Is it, then, a bonfire just kindled?" I questioned. I watched to see whether it would spread: but no; as it did not diminish, so it did not enlarge. "It may be a candle in a house," I then conjectured; "but if so, I can never reach it. It is much too far away: and were it within a yard of me, what would it avail? I should but knock at the door to have it shut in my face." And I sank down where I stood, and hid my face against the ground. I lay still a while: the night-wind swept over the hill and over me, and died moaning in the distance; the rain fell fast, wetting me afresh to the skin. Could I but have stiffened to the still frost--the friendly numbness of death--it might have pelted on; I should not have felt it; but my yet living flesh shuddered at its chilling influence. I rose ere long. The light was yet there, shining dim but constant through the rain. I tried to walk again: I dragged my exhausted limbs slowly towards it. It led me aslant over the hill, through a wide bog, which would have been impassable in winter, and was splashy and shaking even now, in the height of summer. Here I fell twice; but as often I rose and rallied my faculties. This light was my forlorn hope: I must gain it. Having crossed the marsh, I saw a trace of white over the moor. I approached it; it was a road or a track: it led straight up to the light, which now beamed from a sort of knoll, amidst a clump of trees--firs, apparently, from what I could distinguish of the character of their forms and foliage through the gloom. My star vanished as I drew near: some obstacle had intervened between me and it. I put out my hand to feel the dark mass before me: I discriminated the rough stones of a low wall--above it, something like palisades, and within, a high and prickly hedge. I groped on. Again a whitish object gleamed before me: it was a gate--a wicket; it moved on its hinges as I touched it. On each side stood a sable bush-holly or yew. Entering the gate and passing the shrubs, the silhouette of a house rose to view, black, low, and rather long; but the guiding light shone nowhere. All was obscurity. Were the inmates retired to rest? I feared it must be so. In seeking the door, I turned an angle: there shot out the friendly gleam again, from the lozenged panes of a very small latticed window, within a foot of the ground, made still smaller by the growth of ivy or some other creeping plant, whose leaves clustered thick over the portion of the house wall in which it was set. The aperture was so screened and narrow, that curtain or shutter had been deemed unnecessary; and when I stooped down and put aside the spray of foliage shooting over it, I could see all within. I could see clearly a room with a sanded floor, clean scoured; a dresser of walnut, with pewter plates ranged in rows, reflecting the redness and radiance of a glowing peat-fire. I could see a clock, a white deal table, some chairs. The candle, whose ray had been my beacon, burnt on the table; and by its light an elderly woman, somewhat rough-looking, but scrupulously clean, like all about her, was knitting a stocking. I noticed these objects cursorily only--in them there was nothing extraordinary. A group of more interest appeared near the hearth, sitting still amidst the rosy peace and warmth suffusing it. Two young, graceful women--ladies in every point--sat, one in a low rocking-chair, the other on a lower stool; both wore deep mourning of crape and bombazeen, which sombre garb singularly set off very fair necks and faces: a large old pointer dog rested its massive head on the knee of one girl--in the lap of the other was cushioned a black cat. A strange place was this humble kitchen for such occupants! Who were they? They could not be the daughters of the elderly person at the table; for she looked like a rustic, and they were all delicacy and cultivation. I had nowhere seen such faces as theirs: and yet, as I gazed on them, I seemed intimate with every lineament. I cannot call them handsome--they were too pale and grave for the word: as they each bent over a book, they looked thoughtful almost to severity. A stand between them supported a second candle and two great volumes, to which they frequently referred, comparing them, seemingly, with the smaller books they held in their hands, like people consulting a dictionary to aid them in the task of translation. This scene was as silent as if all the figures had been shadows and the firelit apartment a picture: so hushed was it, I could hear the cinders fall from the grate, the clock tick in its obscure corner; and I even fancied I could distinguish the click-click of the woman's knitting-needles. When, therefore, a voice broke the strange stillness at last, it was audible enough to me. "Listen, Diana," said one of the absorbed students; "Franz and old Daniel are together in the night-time, and Franz is telling a dream from which he has awakened in terror--listen!" And in a low voice she read something, of which not one word was intelligible to me; for it was in an unknown tongue--neither French nor Latin. Whether it were Greek or German I could not tell. "That is strong," she said, when she had finished: "I relish it." The other girl, who had lifted her head to listen to her sister, repeated, while she gazed at the fire, a line of what had been read. At a later day, I knew the language and the book; therefore, I will here quote the line: though, when I first heard it, it was only like a stroke on sounding brass to me--conveying no meaning:-- "'Da trat hervor Einer, anzusehen wie die Sternen Nacht.' Good! good!" she exclaimed, while her dark and deep eye sparkled. "There you have a dim and mighty archangel fitly set before you! The line is worth a hundred pages of fustian. 'Ich wage die Gedanken in der Schale meines Zornes und die Werke mit dem Gewichte meines Grimms.' I like it!" Both were again silent. "Is there ony country where they talk i' that way?" asked the old woman, looking up from her knitting. "Yes, Hannah--a far larger country than England, where they talk in no other way." "Well, for sure case, I knawn't how they can understand t' one t'other: and if either o' ye went there, ye could tell what they said, I guess?" "We could probably tell something of what they said, but not all--for we are not as clever as you think us, Hannah. We don't speak German, and we cannot read it without a dictionary to help us." "And what good does it do you?" "We mean to teach it some time--or at least the elements, as they say; and then we shall get more money than we do now." "Varry like: but give ower studying; ye've done enough for to-night." "I think we have: at least I'm tired. Mary, are you?" "Mortally: after all, it's tough work fagging away at a language with no master but a lexicon." "It is, especially such a language as this crabbed but glorious Deutsch. I wonder when St. John will come home." "Surely he will not be long now: it is just ten (looking at a little gold watch she drew from her girdle). It rains fast, Hannah: will you have the goodness to look at the fire in the parlour?" The woman rose: she opened a door, through which I dimly saw a passage: soon I heard her stir a fire in an inner room; she presently came back. "Ah, childer!" said she, "it fair troubles me to go into yond' room now: it looks so lonesome wi' the chair empty and set back in a corner." She wiped her eyes with her apron: the two girls, grave before, looked sad now. "But he is in a better place," continued Hannah: "we shouldn't wish him here again. And then, nobody need to have a quieter death nor he had." "You say he never mentioned us?" inquired one of the ladies. "He hadn't time, bairn: he was gone in a minute, was your father. He had been a bit ailing like the day before, but naught to signify; and when Mr. St. John asked if he would like either o' ye to be sent for, he fair laughed at him. He began again with a bit of a heaviness in his head the next day--that is, a fortnight sin'--and he went to sleep and niver wakened: he wor a'most stark when your brother went into t' chamber and fand him. Ah, childer! that's t' last o' t' old stock--for ye and Mr. St. John is like of different soart to them 'at's gone; for all your mother wor mich i' your way, and a'most as book-learned. She wor the pictur' o' ye, Mary: Diana is more like your father." I thought them so similar I could not tell where the old servant (for such I now concluded her to be) saw the difference. Both were fair complexioned and slenderly made; both possessed faces full of distinction and intelligence. One, to be sure, had hair a shade darker than the other, and there was a difference in their style of wearing it; Mary's pale brown locks were parted and braided smooth: Diana's duskier tresses covered her neck with thick curls. The clock struck ten. "Ye'll want your supper, I am sure," observed Hannah; "and so will Mr. St. John when he comes in." And she proceeded to prepare the meal. The ladies rose; they seemed about to withdraw to the parlour. Till this moment, I had been so intent on watching them, their appearance and conversation had excited in me so keen an interest, I had half-forgotten my own wretched position: now it recurred to me. More desolate, more desperate than ever, it seemed from contrast. And how impossible did it appear to touch the inmates of this house with concern on my behalf; to make them believe in the truth of my wants and woes--to induce them to vouchsafe a rest for my wanderings! As I groped out the door, and knocked at it hesitatingly, I felt that last idea to be a mere chimera. Hannah opened. "What do you want?" she inquired, in a voice of surprise, as she surveyed me by the light of the candle she held. "May I speak to your mistresses?" I said. "You had better tell me what you have to say to them. Where do you come from?" "I am a stranger." "What is your business here at this hour?" "I want a night's shelter in an out-house or anywhere, and a morsel of bread to eat." Distrust, the very feeling I dreaded, appeared in Hannah's face. "I'll give you a piece of bread," she said, after a pause; "but we can't take in a vagrant to lodge. It isn't likely." "Do let me speak to your mistresses." "No, not I. What can they do for you? You should not be roving about now; it looks very ill." "But where shall I go if you drive me away? What shall I do?" "Oh, I'll warrant you know where to go and what to do. Mind you don't do wrong, that's all. Here is a penny; now go--" "A penny cannot feed me, and I have no strength to go farther. Don't shut the door:--oh, don't, for God's sake!" "I must; the rain is driving in--" "Tell the young ladies. Let me see them--" "Indeed, I will not. You are not what you ought to be, or you wouldn't make such a noise. Move off." "But I must die if I am turned away." "Not you. I'm fear'd you have some ill plans agate, that bring you about folk's houses at this time o' night. If you've any followers--housebreakers or such like--anywhere near, you may tell them we are not by ourselves in the house; we have a gentleman, and dogs, and guns." Here the honest but inflexible servant clapped the door to and bolted it within. This was the climax. A pang of exquisite suffering--a throe of true despair--rent and heaved my heart. Worn out, indeed, I was; not another step could I stir. I sank on the wet doorstep: I groaned--I wrung my hands--I wept in utter anguish. Oh, this spectre of death! Oh, this last hour, approaching in such horror! Alas, this isolation--this banishment from my kind! Not only the anchor of hope, but the footing of fortitude was gone--at least for a moment; but the last I soon endeavoured to regain. "I can but die," I said, "and I believe in God. Let me try to wait His will in silence." These words I not only thought, but uttered; and thrusting back all my misery into my heart, I made an effort to compel it to remain there--dumb and still. "All men must die," said a voice quite close at hand; "but all are not condemned to meet a lingering and premature doom, such as yours would be if you perished here of want." "Who or what speaks?" I asked, terrified at the unexpected sound, and incapable now of deriving from any occurrence a hope of aid. A form was near--what form, the pitch-dark night and my enfeebled vision prevented me from distinguishing. With a loud long knock, the new-comer appealed to the door. "Is it you, Mr. St. John?" cried Hannah. "Yes--yes; open quickly." "Well, how wet and cold you must be, such a wild night as it is! Come in--your sisters are quite uneasy about you, and I believe there are bad folks about. There has been a beggar-woman--I declare she is not gone yet!--laid down there. Get up! for shame! Move off, I say!" "Hush, Hannah! I have a word to say to the woman. You have done your duty in excluding, now let me do mine in admitting her. I was near, and listened to both you and her. I think this is a peculiar case--I must at least examine into it. Young woman, rise, and pass before me into the house." {Hush, Hannah; I have a word to say to the woman: p323.jpg} With difficulty I obeyed him. Presently I stood within that clean, bright kitchen--on the very hearth--trembling, sickening; conscious of an aspect in the last degree ghastly, wild, and weather-beaten. The two ladies, their brother, Mr. St. John, the old servant, were all gazing at me. "St. John, who is it?" I heard one ask. "I cannot tell: I found her at the door," was the reply. "She does look white," said Hannah. "As white as clay or death," was responded. "She will fall: let her sit." And indeed my head swam: I dropped, but a chair received me. I still possessed my senses, though just now I could not speak. "Perhaps a little water would restore her. Hannah, fetch some. But she is worn to nothing. How very thin, and how very bloodless!" "A mere spectre!" "Is she ill, or only famished?" "Famished, I think. Hannah, is that milk? Give it me, and a piece of bread." Diana (I knew her by the long curls which I saw drooping between me and the fire as she bent over me) broke some bread, dipped it in milk, and put it to my lips. Her face was near mine: I saw there was pity in it, and I felt sympathy in her hurried breathing. In her simple words, too, the same balm-like emotion spoke: "Try to eat." "Yes--try," repeated Mary gently; and Mary's hand removed my sodden bonnet and lifted my head. I tasted what they offered me: feebly at first, eagerly soon. "Not too much at first--restrain her," said the brother; "she has had enough." And he withdrew the cup of milk and the plate of bread. "A little more, St. John--look at the avidity in her eyes." "No more at present, sister. Try if she can speak now--ask her her name." I felt I could speak, and I answered--"My name is Jane Elliott." Anxious as ever to avoid discovery, I had before resolved to assume an _alias_. "And where do you live? Where are your friends?" I was silent. "Can we send for any one you know?" I shook my head. "What account can you give of yourself?" Somehow, now that I had once crossed the threshold of this house, and once was brought face to face with its owners, I felt no longer outcast, vagrant, and disowned by the wide world. I dared to put off the mendicant--to resume my natural manner and character. I began once more to know myself; and when Mr. St. John demanded an account--which at present I was far too weak to render--I said after a brief pause-- "Sir, I can give you no details to-night." "But what, then," said he, "do you expect me to do for you?" "Nothing," I replied. My strength sufficed for but short answers. Diana took the word-- "Do you mean," she asked, "that we have now given you what aid you require? and that we may dismiss you to the moor and the rainy night?" I looked at her. She had, I thought, a remarkable countenance, instinct both with power and goodness. I took sudden courage. Answering her compassionate gaze with a smile, I said--"I will trust you. If I were a masterless and stray dog, I know that you would not turn me from your hearth to-night: as it is, I really have no fear. Do with me and for me as you like; but excuse me from much discourse--my breath is short--I feel a spasm when I speak." All three surveyed me, and all three were silent. "Hannah," said Mr. St. John, at last, "let her sit there at present, and ask her no questions; in ten minutes more, give her the remainder of that milk and bread. Mary and Diana, let us go into the parlour and talk the matter over." They withdrew. Very soon one of the ladies returned--I could not tell which. A kind of pleasant stupor was stealing over me as I sat by the genial fire. In an undertone she gave some directions to Hannah. Ere long, with the servant's aid, I contrived to mount a staircase; my dripping clothes were removed; soon a warm, dry bed received me. I thanked God--experienced amidst unutterable exhaustion a glow of grateful joy--and slept. | Summary: After two days, the coachman leaves Jane at Whitcross. She spends the night on the moors. The next day she tries to find work or food but finds that no one is ready to help her. She is forced to spend the next night also in the open, and the weather turns cold and wet. She tries to meet the clergyman, but he is away on account of his father's death. Except for a piece of bread from a farmer, and some cold porridge offered by a little girl, she cannot find much to eat. She almost resigns herself to death. It is then that she sees a distant light from a lonely house, towards which she starts walking. On reaching the house, she looks in through a window. She sees two sisters, Diana and Mary, studying German. Their servant, Hannah, is knitting. Hannah refuses to let Jane in. Out of exhaustion Jane collapses at the entrance of the house. Their brother, the clergyman St. John Rivers, arrives just then and insists upon Jane's being let in. She is given food and drink and put to bed. She gives her name as Jane Elliot and asks not to be questioned further until she feels better. | 9,767 | 266 | booksum | en |
Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a handle for ball striking equipment (such as a golf club), and more particularly to a handle that comes with a gripping mechanism to fit an underlisting for both left-handed and right-handed users. 2. Description of the Related Art In general, a traditional ball striking equipment such as a tennis racket, a badminton racket, a golf club or a hockey stick comes with a handle wound with a wrapping strap that is made of genuine leather or artificial leather for users to grip. The traditional wrapping strap usually has through holes, pressing patterns, or polished structures, and an elastic member is buried into or added to the wrapping strap to provide the functions of absorbing sweats, resisting slippery and reducing vibrations, so that a user's hands can firmly and comfortably grip the ball striking equipment. Regardless of the design of the wrapping straps, the overall mechanical properties such as the sweat absorbing capability, the coefficient of friction, the elasticity, and the shock resistance are substantially the same, and thus the traditional wrapping strap cannot provide different grips to fit different positions of a user's hand. As to the sports that require both hands to play, such as gripping a golf club or a tennis racket, the hand that grips the rear end of a club or a racket is the main source of force for striking the ball and thus bearing a vast majority of the reaction from the action of striking balls. The other hand that holds the front end of the club or racket primarily guides the striking direction. In other words, the position of the hand that drives the club or racket should be given better effects of absorbing shocks and resisting slippery, and the position of the other hand that guides the striking direction should be given a more comfortable grip, since both hands have different functions. SUMMARY OF THE INVENTION The present invention provides a solution for gripping a ball striking equipment by both hands and a gripping mechanism to fit the requirements of both left-handed and right-handed users. The technical solution provided by the present invention comprises the following elements: An underlisting is provided for passing a handle rod of a ball striking equipment inside, and the outside has a first end, a second end, a first section defined by extending a predetermined distance from the first end to the second end, and a second section defined by extending from a terminating end of the first section to the second end, and the overall external diameter of the second section is smaller than the terminating end of the first section, and thus an external diameter difference is occurred at the boundary of the starting end of the second section and the terminating end of the first section. A wrapping strap is fixed outside the second section. Therefore, it is a primary objective of the present invention to divide the wrapping strap and the first section of the underlisting into two gripping sections, wherein the first section provides better shock absorbing and slippery resisting effects, and the wrapping strap provides a soft and comfortable slippery resisting effect. Another objective of the present invention is to provide a first section for the hand that gives the main source of force to strike a ball, and the wrapping strap for the other hand that controls the striking direction, when both hands grip a handle. As a result, the handle can provide different gripping mechanisms for both hands of the user. A further objective of the present invention is to provide the first section and the section of the wrapping strap for the grip of both hands, so as to create different visual effects for the sports equipment handle. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a planar view of an underlisting of the invention; FIG. 2 is a cross-sectional view of a portion of an underlisting of the invention; FIG. 3 is a schematic view of an underlisting wound by a genuine leather wrapping strap according to the invention; FIG. 4 is a schematic view of an underlisting wound by a genuine leather wrapping strap and two fixing objects used for fixing the starting end and the terminating end of the wrapping strap according to the invention; FIG. 5 is a perspective view of an underlisting and a genuine leather wrapping strap forming a ball striking equipment handle according to the invention; FIG. 6 is a cross-sectional view of Section 6 - 6 as depicted in FIG. 5 ; FIG. 7 is a cross-sectional view of Section 7 - 7 as depicted in FIG. 5 ; FIG. 8 is a perspective view of an underlisting and another genuine leather wrapping strap forming a ball striking equipment handle according to the invention; FIG. 9 is a cross-sectional view of Section 9 - 9 as depicted in FIG. 8 ; and FIG. 10 is a cross-sectional view of a portion of an underlisting and a genuine leather wrapping strap according to the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Refer to FIG. 1 for the planar view of an underlisting 10 of the present invention. The underlisting 10 is a soft elastic material, and its interior is capable of accommodating a handle rod of a ball striking equipment such as a golf club, and its exterior has a first end 11, a second end 12 and a conical profile formed between the first and second ends 11, 12. A first section 13 is defined by extending a predetermined distance from the first end 11 along the direction of the conical profile to the second end 12, and a second section 14 is defined by extending from a terminating end 131 of the first section 13 along the direction of the conical profile to the second end 12, and the overall external diameter of the second section 14 is smaller than the terminating end 131 of the first section 13. Therefore, an external diameter difference 15 is occurred at the boundary of the starting end 141 of the second section 14 and the terminating end 131 of the first section 13. Further, a protruded ring 16 is disposed on the second section 14 at a predetermined distance proximate to the second end 12, and a ring groove 17 is formed between the protruded ring 16 and the second end 12. Refer to FIG. 2 for the cross-sectional view of a portion of the underlisting 10. In FIG. 2, the external diameter difference 15 is used to form an inwardly inclined surface 132 around the terminating end 131 of the first section 13, such that a circular corner 133 is formed at the boundary of the first section 13 and the second section 14. Refer to FIG. 3 for the schematic view of the underlisting 10 wound with a wrapping strap 20. Basically, the wrapping strap 20 could be any traditional wrapping strap spirally wound around the second section 14. Referring to FIGS. 4 to 6, a starting end 201 wound with the wrapping strap 20 is extended into the round corner 133 and wound spirally such that the corresponding edges are overlapped with each other, and the overlapped portion 203 is not protruded from an external profile 204 wound by the wrapping strap 20, and the winding at a terminating end 202 is evenly ended at the protruded ring 16. The wrapping strap 20 is fixed onto the terminating end 202 of the winding by a fixing member 31 to prevent the terminating end 202 from becoming loosened. Refer to FIG. 7, the fixing member 31 at the boundary of the terminating end 202 of the wrapping strap 20 and the protruded ring 16 is an elastic ring with an elastic bidning force, and the fixing member 31 provides a binding force to the terminating end 202 of the wrapping strap 20 and the protruded ring 16. A fixing section 311 protruded from a wall of the fixing member 31 is embedded into the ring groove 17, so that the fixing member 31 can be secured at a predetermined position. Refer to FIGS. 8 and 9 for the schematic view of the underlisting 10 being wound by a wrapping strap in another fashion. The wrapping strap 21 comprised of a first and a second strap bodies 211, 212 with different widths spirally wind the second section 14. Similarly to the description above, the starting end 213 of the winding of the wrapping strap 21 is extended into the circular corner 133 and spirally wound, and the corresponding edges are overlapped with each other. The overlapped portion 214 is not protruded from the external profile 215 of the wrapping strap 20. The terminating end 216 is evenly terminated at the protruded ring 16. The wound wrapping strap 21 is fixed at the position of the by a fixing member 216 with a binding force. The wrapping strap 20, 21 as illustrated in FIGS. 1 to 9 is a long bar structure made of genuine leather. Refer to FIG. 10 for the cross-sectional view of a section of the underlisting 10 being wound by a wrapping strap in another fashion. In FIG. 10, the wrapping strap 22 is a long bar structure made of artificial leather. Similar to the description above, the starting end 221 of the winding of the wrapping strap 22 is extended into the circular corner 133 and spirally wound, and the corresponding edges are overlapped with each other. The overlapped portion 222 is not protruded from the external profile 223 of the wrapping strap 20. The foregoing first section 12 and the wrapping strap 20, 21, 22 divide the underlisting 10 into upper and lower gripping sections. The first section 13 has an external profile for an even grip, and the wrapping strap 20, 21, 22 has a wavy external profile. For example, a right-handed golf player uses his/her left hand that gives the main source of striking force to grip the first section 13 and his/her right that controls the striking direction of the ball to grip the wrapping strap 20, 21, 22. Since the wrapping strap 20, 21, 22 has a spiral wavy external profile, therefore it provides a better slippery-resisting effect and a more comfortable grip, and the player's right hand can control the striking direction more stably. The first section 13 uses the thickness and elasticity of its own structure to provide a better shock resistance and reduce the reaction from the striking ball on the left hand. The rubber material of the underlisting 10 and the slippery-proof pattern on the underlisting give more frictions and provide a better slippery resisting effect, so that the player can securely grip the handle and control the force of striking the ball. The handle of the present invention can simultaneously provide a gripping mechanism for both left-handed and right-handed players. To fit the palm size and the gripping position of different players, and simultaneously provide the gripping mechanism simultaneously for both left-handed and right-handed players, the ratio of the axial lengths of the first section 13 and the second section 14 of the underlisting 10 can be changed. While the invention has been described by way of example and in terms of a preferred embodiment, it is to be understood that the invention is not limited thereto. To the contrary, it is intended to cover various modifications and similar arrangements and procedures, and the scope of the appended claims therefore should be accorded the broadest interpretation so as to encompass all such modifications and similar arrangements and procedures. | Summary: A handle structure for ball striking equipment includes an underlisting for passing a handle rod of a ball striking equipment through its interior and having a first section and a second section at its exterior, a wrapping strap fixed on the outside of the second section, and the wrapping strap and the first section of the underlisting divide a handle holding section into two gripping sections, and the first section gives a shock absorbing and a slippery resisting effects, and the wrapping strap provides a soft, comfortable and slippery-proof gripping effect. | 2,783 | 114 | big_patent | en |
Summarize: By. Lizzie Parry. An easyJet flight was grounded for two hours after a security alert was sparked when schoolchildren reported seeing a man writing Arabic in a notebook. The students, who were aged 15 and 16, alerted cabin crew after spotting the man writing in what appeared to be Arabic script, while waiting for take off. The flight from Amsterdam to Newcastle was delayed to allow for 16 of the 45-strong group of students, believed to be from a school in Northumberland, to get off the aircraft. Delayed: An easyJet flight from Amsterdam to Newcastle was delayed for two hours after a group of school children reported seeing another passenger writing in what appeared to be Arabic script. Technical support worker Adam Robson, 21, who was sat next to the man, said he first became aware of the issue when he felt kicking and banging on the back of his seat as one of the student's suffered a panic attack. He said: 'The guy had a notebook and people were looking over at it as it had both Arabic writing and English words. 'One of the students called a flight attendant over and pointed it out,' said Mr Robson, from Newcastle’s West End, who had been on a romantic weekend away with his girlfriend Lauren. 'She went away, but just as we were about to take off there was a kicking and banging on my seat from behind us and the student was having a panic attack. 'He went from the window seat into the aisle and ran to the back of the plane. 'The next think we know the pilot has come on and said for safety reasons we were going back to the terminal.' Mr Robson said he and other passengers were kept aboard the aircraft as a number of the pupils and their luggage were taken off the aircraft - as temperatures in the cabin soared to 30 degrees. 'The attendants had to bring us cups of cold water because it was so hot,' he said. Genuine: Passenger Adam Robson, who was sitting next to the man on the flight which took off from Amsterdam's Schiphol airport, said he 'was harmless'. He said the man was an Iranian masters student at Teesside University and had been in Amsterdam visiting his wife. The flight, on Monday, eventually took off, with Mr Robson chatting to the man, who he said seemed 'completely harmless'. 'It turns out he was an Iranian masters student at Teesside University and the reason he was in Amsterdam was because his wife was studying there as she’d been unable to transfer to a North East university. 'He makes the trips regularly and he said he always carries his notebook, which he showed me. It had some Arabic, but the further through you went the more English it had as he had written down sections of newspapers and was using it to teach himself more of the language. 'He was really genuine. 'Thankfully he wasn’t aware of it and he didn’t understand what was going on.' On arriving back in Newcastle Adam said he helped the man, whose name he did not know, make his way to Central Station so he could catch a train back to his home in Middlesbrough. The pupils who got off the flight returned on another easyJet flight which landed at Stansted yesterday. An easyJet spokesman said: 'easyJet can confirm that flight EZY6554 from Amsterdam to Newcastle was delayed while a number of passengers from a school group chose to move onto an alternative flight. 'This was due to a nervous young passenger in the group who raised a security issue prior to take off. The Captain fully assessed the issue and the flight was cleared for take off by easyJet’s security department. 'The safety and wellbeing of our passengers is always easyJet’s priority. 'The flight was delayed by two hours 20 minutes while the issue was investigated and to enable 16 members of the group to be offloaded along with their baggage. 'easyJet transferred the passengers onto an alternative flight of charge free of charge. We would like to thank passengers for their understanding and apologise for any inconvenience the delay may have caused them.' Persian, known to native speakers as Farsi, is the official language of Iran, parts of Afghanistan and Tajikistan. The Persian of Iran is written in a joined-up Arabic script that can be highly ornamental. Minority populations in other Middle Eastern countries - Bahrain, Iraq, Oman, Yemen and the United Arab Emirates - also speak Persian, as do communities in Europe, Turkey, Australia, the USA and Canada | Summary: Amsterdam to Newcastle flight grounded after security alert on Monday. Group of students told cabin crew they had seen a fellow passenger writing in what appeared to be Arabic script. Adam Robson, 21, was sitting next to the man, who was 'harmless' 16 of the students, aged 15 to 16 got off the flight, delaying it by two hours. Mr Robson said the man was an Iranian masters student at Teesside University who had been visiting his wife. 'He was really genuine. Thankfully... he didn't understand what was going on,' said Mr Robson. | 988 | 135 | cnn_dailymail | en |
Write a title and summarize: Tumorigenesis is a multi-step process in which normal cells transform into malignant tumors following the accumulation of genetic mutations that enable them to evade the growth control checkpoints that would normally suppress their growth or result in apoptosis. It is therefore important to identify those combinations of mutations that collaborate in cancer development and progression. DNA copy number alterations (CNAs) are one of the ways in which cancer genes are deregulated in tumor cells. We hypothesized that synergistic interactions between cancer genes might be identified by looking for regions of co-occurring gain and/or loss. To this end we developed a scoring framework to separate truly co-occurring aberrations from passenger mutations and dominant single signals present in the data. The resulting regions of high co-occurrence can be investigated for between-region functional interactions. Analysis of high-resolution DNA copy number data from a panel of 95 hematological tumor cell lines correctly identified co-occurring recombinations at the T-cell receptor and immunoglobulin loci in T- and B-cell malignancies, respectively, showing that we can recover truly co-occurring genomic alterations. In addition, our analysis revealed networks of co-occurring genomic losses and gains that are enriched for cancer genes. These networks are also highly enriched for functional relationships between genes. We further examine sub-networks of these networks, core networks, which contain many known cancer genes. The core network for co-occurring DNA losses we find seems to be independent of the canonical cancer genes within the network. Our findings suggest that large-scale, low-intensity copy number alterations may be an important feature of cancer development or maintenance by affecting gene dosage of a large interconnected network of functionally related genes. Tumor development is generally thought to be a process in which healthy cells transform into malignant tumor cells through the step-wise acquisition of oncogenic alterations [1], [2]. This implies that certain changes have to occur together for effective oncogenic transformation of a normal cell. There are a multitude of (epi-) genetic lesions that cause deregulated expression of oncogenes and tumor suppressor genes. Co-operative deregulation of cancer genes has indeed been observed in several different settings. Retroviral insertional mutagenesis screens in mice have shown preferential co-mutation of specific combinations of genes within the same tumor [3]. Likewise, in a study where a thousand individual tumors were screened for mutations in 17 different oncogenes, preferential co-mutation of the PIK3CA and KRAS genes was observed [4]. Besides single basepair mutations or retroviral integrations, the activity of genes can also be perturbed by DNA copy number alterations that arise as a result of genomic instability, which is frequently observed in tumor cells [1]. Whether genomic instability is important for tumor initiation is controversial, but its contribution to tumor progression is undisputed [5], [6]. Loss of DNA is a mechanism for the tumor to eliminate copies of tumor suppressor genes, which prevent cancer formation. Conversely, DNA copy number gain or amplification may lead to activation of oncogenes that promote tumor development. We aimed to find genomic regions of gains and losses that are preferentially gained or lost together. We could subsequently link genes that lie in co-occurring regions to each other, allowing us to find functional interactions that reveal the mechanisms underlying tumor development. DNA copy number alterations (CNAs) may be measured on microarray platforms [7]. Array-based comparative genomic hybridization (aCGH) of differentially labeled tumor and normal (2n) DNA is performed on oligonucleotide- or Bacterial Artificial Chromosome (BAC) based microarray platforms. For each probe on the microarray, the ratio of signal intensities of tumor versus normal DNA is a measure of the relative DNA copy number of the corresponding genomic region in the tumor sample. Platforms designed to identify single nucleotide polymorphisms (SNPs) can also infer CNAs by comparing the raw probe intensity values measured in a tumor sample with a reference sample. In order to extract those DNA copy number aberrations that preferentially occur together, we developed an analysis framework. The basic premise of our analysis is to define a pair-wise score for any given pair of genomic locations present in the dataset. This scoring index will only be high if both genomic locations are recurrently aberrated in multiple independent samples within the tumor panel, and if they co-vary similarly over the different samples (Figure 1). Using a Gaussian kernel convolution method we look for aggregates of high scores in the 2D genomic pair-wise space (Figure 2). The top peaks in the convolved score matrix can be mapped back to two distinct co-mutated genomic locations. The genes that reside in these genomic locations can then be functionally related to each other. The raw data consist of non-discrete measurements of the average DNA copy number of the population of cells present in the measured sample. The signal consists of a measurement of a heterogeneous population of tumor cells, which may contain many populations potentially carrying different mutations and copy number alterations, as well as normal (diploid) cells. To reduce heterogeneity as much as possible we choose to analyze a collection of hematopoietic tumor cell lines, which on a per-sample basis can be considered clonal. There were several other reasons for analyzing this particular dataset. First, it is a high resolution dataset of well-characterized, clinically relevant samples. Although these samples are cell lines, they are widely used as a model system for the diseases from which they have been derived. Second, this collection of samples includes cell lines derived from T- and B-cell leukemias carrying rearranged T-cell receptor and immunoglobulin loci, respectively. We therefore should be able to separate these two distinct lymphoid malignancies based on co-occurring DNA copy number losses at the T-cell receptor and immunoglobulin loci. During T- and B-cell development, these loci undergo DNA recombination and gene deletion in a process known as V (D) J-recombination. The human genome contains three specific T-cell receptor loci (alpha/delta, beta and gamma) on two different chromosomes that determine their variability. B-cells have three different loci (IgG kappa, IgG lambda and the IgG heavy chain) on three different chromosomes that undergo recombination to generate a diverse repertoire of immunoglobulins. Since T- and B-cells only undergo recombination of their respective loci after lineage commitment, it is unlikely that T-cell receptor loci are recombined in B-cells and vice-versa. If our approach is successful at finding co-occurring losses, it should identify the co-occurring rearrangements at the T-cell receptor alpha/delta and beta/gamma loci in T-cell leukemias. Similarly, we should be able to pick up co-occurring losses at the IgG kappa, lambda and heavy chain loci in B-cell malignancies. A classic example of finding associations in a large (binary) dataset is association rule mining. Identification of cooperating events in continuous data requires a different approach than binary association rule mining. First we developed a method to score for co-occurrence between two continuous measurements (Figure 1). We then applied this score in a framework that is able to find co-occurrences in genome-wide measurements. This framework is shown in Figure 2 and is detailed in the Materials and Methods. DNA copy number measurements at two different genomic loci can be visualized in a 2D space, with each axis representing measurements at a certain genomic locus. A point in this space represents a sample in which both loci were measured. Figure 1a shows four hypothetical combinations of measurements. We sought to score for co-occurring high or low values in the DNA copy number data; in other words, regions that display similar patterns of large-amplitude amplification and deletion across the tumor set. This situation is shown in the third panel Figure 1a. The other panels show other potential situations that may arise when comparing two continuous measurements. To score for co-occurring gains all negative values are set to zero (Figure 1b). To score for co-occurring losses all positive values need to be set to zero and the absolute values of the measurements need to be used. We use the covariance of the two measurements to score for co-occurring loci. This score only rewards a high value to a truly co-occurring and co-varying pairs of measurements (Figure 1c, right panel). However, a high covariance alone is not sufficient, since it is possible that a high covariance occurs while at least one of the loci never reaches a high amplitude (see Figures 1e and 1f). For this reason we multiply the covariance score with the sum of the individual valued in each sample. This method of scoring only rewards a high value to a co-varying pair of measurements with a large aberration amplitude across the tumors (Figure 1c, right panel). The co-occurrence scores can be computed for every pair of genomic loci (Figure 2c). By performing a two-dimensional Gaussian kernel convolution on these scores in the co-occurrence space we can take local neighborhood effects into account. This operation is performed for different kernel widths in order to capture scale dependent effects, resulting in a Convolved Co-occurrence Matrix (CCM) as shown in Figure 2d. High values in this matrix represent candidate co-occurring regions in the data. A peak in the CCM can be mapped back to two specific loci, the size of which is determined by the σ parameter of the Gaussian function used to convolve the score matrix (Figure 2e). The genes that are located in the loci associated with a peak in the CCM are subsequently investigated. We examined both enrichment for known cancer genes in these gene lists and we investigated functional relationships between the genes derived from the two loci (Figure 2f). Additional details can be found in the Materials and Methods section. We ran our analyses on the aCGH profiles of 95 hematological tumor cell lines analyzed on the Affymetrix Genome-Wide Human SNP Array 6. 0. See the supplemental data (Dataset S1) for a list of the cell lines that were analyzed. The data was generated by the Cancer Genome Project (Wellcome Trust Sanger Institute, Hinxton, UK). We employed three scale parameters: 2Mb (σ = 1/3 Mb), 10Mb (σ = 5/3 Mb) and 20Mb (σ = 10/3 Mb). In the remainder of this text we will refer to these as Scales 2,10 and 20. These scales roughly determine the size of the aberrant regions we expect to find. By employing a small, medium and large scale we maximize the chance of detecting co-occurring changes of all possible sizes. To remain conservative we limited our primary analysis to the top 50 peaks in the Convolved Co-occurrence Matrix (CCM) for each of the scales and each of the comparisons (gain-gain, loss-loss, loss-gain). This resulted in 9 top-50 lists of co-occurring regions retrieved from this dataset. A substantial fraction of the 95 cell lines are derived from T- or B-cell lymphomas with functionally rearranged T-cell receptor or IgG genes. We therefore expected to identify co-occurring losses at the T-cell receptor alpha/delta and beta/gamma loci in the T-cell leukemias. Similarly, our method should identify co-occurring losses at the IgG kappa, lambda and heavy chain loci in B-cell malignancies. Because the recombination loci for both the T-cell receptor and the IgG genes are both relatively small (in the 1Mb range) we expected to retrieve these co-occurring losses in the small (2 Mb) scale analyses. Since we disregarded co-occurrences on the same chromosome we expected to find five co-occurring losses. Indeed, four of the five expected co-occurring losses are present in the top 50 peaks of the Scale 2 analyses (Table 1). Figure 3 shows two examples from the top 50 lists of co-occurring loci. The separation of T- and B-cell lines is immediately apparent. T-cell lines are strongly associated with losses in the T-cell receptor loci. A large subset of B-cell lines are associated with losses in the IgG loci. However, a subset of the B-cell lines is not associated with any loss of these loci. In this particular subset of lines the IgG loci seem to be gained. It is known that the IgG loci are favorite partners for oncogenic translocations [8]. Whether this is the cause of the amplification of these loci is not known. While the recovery of the V (D) J-related recombination loci as co-occurring losses serves as a positive control for our analysis approach, we are mainly interested in identifying cooperating genes or regions that might play a role in cancer. To see whether the locations we recover are linked to this disease, we analyzed whether the co-occurring genomic loci are enriched for genes known to play a role in cancer. As a reference gene set we used the Cancer Gene Census list [9]. The results of this analysis are shown in Table 2. As can be seen, the co-lost loci are mainly enriched for tumor suppressor genes, and the gain-gain regions for oncogenes. Since one expects loss of tumor suppressors and gain of oncogenes, this is a logical result, further increasing our confidence that our approach identifies truly relevant genomic loci. While finding enrichment for cancer genes is an encouraging result, this does not explain the possible cooperation between two loci. We expect that the co-occurring loss of two regions points to a functional relationship between the constituents of the genomic loci. A co-occurrence between two genomic regions can point to many different kinds of interactions between the genes residing in both regions, e. g. biochemical interactions of the protein products or functional collaboration of two cancer genes in tumorigenesis. We therefore decided to employ interaction data to shed further light on the genes present in the co-occurring regions. We translated the co-occurring pairs of genomic loci to pairs of gene sets, and we investigated the functional relationships of their protein products using the STRING database [10] (version 8. 1). The STRING model weighs functional associations between genes based on several different sources of evidence, among which: biochemical interaction, joint presence in a pathway, high-throughput interaction experiments, text mining and interactions of homologs in other species. To find a functional relationship between two co-occurring regions we looked for a direct interaction in the STRING database between the two gene-sets defined by our co-occurrence analysis. To determine whether the number of observed interactions is significant, we compared the number of direct interactions found between genes located in the top 50 co-occurring regions to a set of randomly chosen pairs of genomic loci. The metric we used to determine significance is the ratio between the number of interacting genes and the total number of genes found on the genomic loci. A p-value for enrichment for direct interactions was calculated using a two-tailed Fisher' s exact test. Results are shown in Figure 4. As can be seen, the only analysis that resulted in an enrichment of functional interactions is Scale 20, for all three situations. We found no enrichment for interacting protein coding genes on Scale 2 (not shown) and Scale 10. Since we evaluated gene sets in a window one-third the size of the analysis-scale we may be under-estimating the size of the co-occurring loci and the larger Scale 20 actually captures the size of the aberrations best. In order to keep control of the complexity, we considered in our co-occurrence analysis only radially symmetric kernels, i. e. Gaussian kernels with diagonal, equal variance covariance matrices. This implies that asymmetric co-occurring regions – where a small locus co-occurs with a large locus – will not be optimally detected. Since an asymmetric co-occurring region typically consists of a series of symmetric co-occurring regions detected on a smaller scale (just like a rectangle can be constructed from a collection of squares), we set out to construct larger co-occurring regions from the results of the smaller scales using a hierarchical clustering approach. For details see Supplemental Figure S1. Briefly, we collected the loci involved in the top 500 co-occurrences of the Scale 2 analysis. This resulted in 1000 genomic loci. For each pair of loci, we calculated the genomic distance in base pairs. The distance between two loci on different chromosome arms was set to a default high value (1 * 108). This resulted in a 1000×1000 distance matrix. On this distance matrix we performed single linkage hierarchical clustering. The resulting dendrogram was cut at 1 * 107 bp (5 kernel widths). The resulting clusters are unique genomic loci and were represented as nodes in a graph. Clusters were then linked if a co-occurrence was found between individual loci of different clusters. These links are represented as edges in a graph. The result of the clustering analysis is shown in Figure 5. As can be seen in Figure 5 we were able to construct a network of co-occurring copy number changes for the gain-gain, loss-loss and gain-loss situations. As expected, the gain-gain and loss-loss networks show enrichment for oncogenes and tumor suppressor genes, respectively. The gain-loss network only shows enrichment for tumor suppressors. The percentage of genes involved in functional interactions between the nodes that are linked in the graph vastly exceeds the functional interaction enrichment found in the single scale 20 Mb analyses. At least 11% of the genes present in the genomic locations - represented by the nodes in the graphs - have high confidence (>0. 9) annotated functional interactions along the edges as revealed by STRING analysis. The thickness of the edges in the graphs shown in Figure 5 indicates how often a co-occurrence was found in the top 500 of the Scale 2 analysis. Several edges were strongly supported by co-occurrences in the top 500. These strongly supported edges were always associated with loci that were ranked high in the co-occurrence list (as indicated by node size). The nodes that are associated with these highly represented edges seem to form an important subgraph. To reveal these subgraphs, we removed all edges supported by less than 5% of the top 500 co-occurrences. For brevity and simplicity we only consider the gain-gain and loss-loss networks. This resulted in the two core networks shown in Figures 6 and 7. The edge thickness of the gain-gain core network shown in Figure 6 represents the number of functional interactions found using the STRING database between genes that map within the loci described by the nodes. To determine the common denominator among the interacting genes, we employed Ingenuity Pathway Analysis (IPA; Ingenuity Systems) to perform a functional enrichment analysis on all genes residing within the gain-gain core network. This revealed strong enrichment for processes involved in cancer (Figure 6b). From Figure 6a it is immediately apparent that most of the functional interactions are found between 1q and 7p/q. If we remove the 1q node from the entire network described in Figure 5 the enrichment for functional interaction drops dramatically (Figure 6c). Therefore, we hypothesize that the co-occurring gain between 1q and 7p/q is the most important effect in the gain-gain analysis in this dataset. This is strengthened by the fact that almost all known oncogenes within the entire network map to 1q, 7p or 7q (Figure 6a). The well-studied canonical oncogene MYC maps to 8q and is not a determining hub in the gene interaction network as constructed by STRING. The loss-loss core network is shown schematically in Figure 7a. A loss of approximately 18 megabases on chromosome 17p appears to be a central hub, which is co-lost with several other genomic loci. These loci show a very high enrichment of genes that interact with 17p, and of the six loci, four contain multiple known tumor suppressor genes. A functional enrichment analysis of all genes residing on loci co-lost with 17p, reveals many cancer-related processes (Figure 7b), suggesting that the interacting genes are most likely also the cancer-relevant genes. If we remove 17p from the original network we see a large decrease in the percentage of genes involved in functional interactions (Figure 7c) confirming the importance of 17p in the loss-loss network. One of the most intensively studied cancer genes, TP53, resides in the 17p locus. Furthermore, the canonical cancer gene RB1 and the CDKN2A/B locus are present in two of its co-lost regions. Since these are well known tumor suppressors, and therefore the subject of thousands of research papers, they might constitute the bulk of the functional relationships in our analysis. To test this hypothesis, we excluded these four genes and repeated the interaction analysis of the core network. As can be seen in Figure 7c, the enrichment is only slightly lower without the canonical genes, suggesting that the functional relationship between the co-occurring losses on 17p and the other loci are driven by other genes. We investigated the remaining 113 interactors for any interesting interactions that might be a target of this collection of co-occurring losses. Within the total network of interactors we found a sub-network centered on the nuclear co-repressor NCOR1 (TRAC1) (Figure 7d). This interaction network included – besides NCOR1 – the peroxisome proliferator-activated receptor alpha (PPARA), the MAPK pathway suppressor GPS2, the nuclear co-activator (and known tumor-suppressor) p300 and a gene of unknown function, CBFA2T3. All interactions found are based on physical binding and co-occurrence in Pubmed abstracts. To see whether we could find more information regarding the putative tumor suppressor function of the different interactors, we tested if we could corroborate our findings with data from a large retroviral insertional mutagenesis (IM) screen where hematopoietic tumors were induced through Murine Leukemia virus (MuLV) infection of wild-type mice or Trp53 or p19-ARF deficient mice [11]. An illustration of the retroviral insertions sites near Cbfa2t3 is shown in Figure 7d. Although Cbfa2t3 was not flagged as a common integration site, several viral integrations near this gene were found. Remarkably, two individual tumors harbored a bi-allelic integration near the transcription start site of Cbfa2t3, suggesting functional inactivation of this candidate tumor suppressor gene. Indeed, bi-allelic integration is thought to be a hallmark of tumor suppressor genes in IM screens [12]. Given that we find this sub-network of interactors in a co-occurring network of DNA copy number losses and the recovery of inactivating insertions in a retroviral IM screen, we conclude that this network might be a putative tumor suppressor network. Several studies have investigated concerted copy number changes in aCGH data. In studies on lung cancer [13] and ovarian cancer [14] the authors performed a post-hoc co-occurrence analysis on genomic locations that were found to be significantly altered in a one-dimensional analysis. A more integrated effort to analyze relations between CNAs in brain cancer was published recently [15]. Although this study scores systematically for co-aberration, it is limited in resolution as it employs cytobands as the genomic unit within which aberrations are scored. Cytobands are relatively arbitrarily determined entities and are quite heterogeneous in size. Furthermore this approach is dependant on converting the continuous-valued copy number data to discrete copy number calls. This results in loss of important information since it removes the possibility of weighting the intensity of a CNA. In contrast, our approach is able to correct for unequal probe distances, enabling us to perform our analysis on a very high (20 kbp) resolution. In addition, our scoring method not only incorporates the sign of the copy number change, but also its intensity and the concomitant CNAs within the immediate genomic neighborhood. The output of our analysis does not include a measure of significance. Constructing a background distribution based on permutations of the DNA copy number data would mean re-running our analysis thousands of times, a task which remains computationally infeasible at this stage. Furthermore, the multiple-testing problem would have to be properly addressed, given that the number of tests is the square of the number of grid points in the 2D space. Due to the complexity of the analysis procedure (minimum operation and kernel smoothing) the definition of an analytical null distribution has remained elusive. Therefore, we have chosen to work with top n results, residing in the extremes of the results distribution, thus minimizing the chance of including false positives. The top n lists allowed us to generate workable results which we have validated extensively with other sources of evidence. While we were able to use a distributed computing solution for our analysis, we were fortunate to have the required computational architecture at our disposal. Since the problem basically consists of repeating the same action many times it could be well-suited to software optimization or a hardware based solution where the most time-consuming actions are handled by a dedicated processing unit. When looking for areas in the 2D pair-wise space highly enriched for co-occurrence scores we convolve this space with a 2D-Gaussian kernel. The sigma parameter of this function is a representation of the size of the aberrations we expect to recover. Currently we make the implicit assumption that the co-occurring aberrations have the same size by using a symmetric kernel for the convolution. This could be relieved by allowing for an asymmetrical (ellipsoid) Gaussian kernel for all combinations of scales used. Clearly, this comes at the cost of increased computational complexity. Here we resolve this issue by concatenation of the results obtained in a small scale. In this way we can recover co-occurring losses of different sizes that give a better enrichment for functional interactions when combined with the single peaks obtained in a higher scale analysis. In our analysis of a set of cell lines derived from hematological malignancies we found enrichment of cancer related genes and functional interactions in co-occurring DNA copy number changes. Our results suggest that tumorigenesis requires elimination of multiple gatekeeper genes and gain of multiple oncogenes as demonstrated by the presence of many functional interactions between the loci in the gain-gain and loss-loss core networks. Haploinsufficiency is a well known characteristic of several tumor suppressor genes, where simple reduction of gene dosage by loss of gene copies at the DNA level can already promote oncogenic transformation [16]. It is conceivable that changes in gene dosage of multiple interconnected genes involved in cancer-related processes such as cell cycle, DNA repair and signaling can also weaken a cells defense against uncontrolled cell proliferation. In this case, heterozygous loss or gain of large genomic regions, such as the ones identified in this study, might effectively sensitize cells to become tumorigenic. We show that the 17p loss and its co-lost regions are highly enriched for functional relationships, which are not fully explained by the presence of the TP53 gene, often thought to be the single target of this deletion [17]–[19]. Although TP53 is no doubt an important target of the DNA copy number loss, our analysis indicates that the concomitant loss of other genes near TP53, as well as co-occurring losses on the other genomic loci may together account for the full tumorigenic effect. Loss of the loci on 17p, 9p, 9q, 13q, 16q and 22q has been reported previously for several types of hematological malignancies represented in our dataset [20]–[23]. The picture that emerges from this analysis of collaborative aberrations is that many of the reported losses collaborate with the frequently occurring 17p loss as a central hub. We don' t recover co-occurring losses among the spoke loci in the core network. This could suggest that the non-17p regions form subsets of co-occurring losses with 17p, whose interconnections themselves do not occur frequently enough in the top 500 co-occurring losses we investigated. Not all of the gene-gene interactions defined by the 17p network involve the well-known canonical cancer genes TP53, RB1 and CDKN2A (INK4a/ARF). We found one sub-network of genes around NCOR1 which might be an example of other tumor suppressor genes that are affected by the concerted loss of these genomic loci. The hub of this interaction network, NCOR1, is a well-known transcriptional co-repressor that associates in a ligand-independent manner with nuclear receptors [24]. It is responsible, together with the closely related factor SMRT, for recruitment of HDAC proteins to the DNA to induce transcriptional silencing. Its role in cancer is not well-established. NCOR1 null mice die in early embryogenesis [25]. A dominant-negative mutant of NCOR1 is known to increase proliferation in hepatocytes [26] and more recently it has been shown that NCOR1 decreases AKT phosphorylation, thus countering its pro-survival signal [27]. It would seem that specific loss - or at least decrease in gene dosage of NCOR1 - might increase proliferation and promote survival. All interactions between NCOR1 and its partner genes (PPARA, GPS2 and CBFA2T3) have been based on co-occurrence in PubMed abstract and true physical binding [28]–[31]. CBFA2T3 is a close relative of ETO, which is a target of the recurrent AML1-ETO translocation that occurs in acute myeloid leukemia. It has been shown that the fusion gene AML1-ETO actually interferes with the CBFA2T3-NCOR1 interaction, and that its oncogenic effect derives from that inhibition [31]. In a retroviral insertional mutagenesis screen in mice, Cbfa2t3 is recurrently targeted by bi-allelic retroviral integrations, which are predicted to cause functional inactivation of Cbfa2t3 [11]. PPARA is a member of the Peroxisome proliferator-activated receptors, and has been implicated in hepatocellular carcinoma development in rodents [32]. Since other members of this family, such as PPARG, exhibit a tumor suppressor-like phenotype, it is possible that PPARA can act as a tumor suppressor in hematological malignancies. GPS2 is a known suppressor of JNK signaling [33], which is one of the constituents of the MAP kinase signaling pathway. Deregulation of this pathway is a well-known phenomenon in cancer [34]. Taken together with the association between NCOR1 and the known tumor suppressor p300, our data suggest a selective advantage for loss of multiple constituents that interact with NCOR1 since they all may have tumor suppressor-like activities. Many studies focus on a single hematological malignancy in which a single combination of aberrations might be important [19], [35], [36]. Since we examine a large panel of samples derived from many different hematological malignancies, our results might not specifically apply to any single type of lymphoma or leukemia. They might hint at more general processes that are important for the tumors to arise and maintain themselves. However, one should not forget that this analysis is based on a panel of cell lines, which may have adapted to in vitro tissue culture conditions by acquiring additional aberrations that are rarely found in real tumors in patients. Furthermore, given the fact that we examine copy number changes it might be worthwhile to analyze a highly genomically unstable tumor type, such as BRCA1/2-related breast cancer. We have developed a method for genome-wide analysis of collaborating DNA copy number changes and their corresponding networks. Using this approach we have identified a loss-loss network centered around a region on human chromosome 17p. This network is highly enriched for functional relationships and hints at a more complex system of tumor suppression in which many different genes are affected simultaneously to induce cancer. We show one example of a sub-network around the nuclear co-repressor NCOR1 that may be a novel network of tumor suppressor genes that are affected by the observed co-occurring losses. The observation that DNA copy number changes may affect gene dosage of larger numbers of cancer-relevant genes deviates from the classical view where mutations in a few (5–7) cancer genes lead to tumor development. Our data support the notion of cancer-related networks or pathways, where multiple collaborating genes are deregulated simultaneously to induce oncogenesis. Such a network view of oncogenesis is an important step towards developing effective drug targets because it increases the number of potential targets. However, this view also implies that multiple molecules need to be targeted simultaneously in order to achieve optimal therapy response and to reduce the risk of therapy resistance. Datasets consisting of array-based copy number measurements are continuously increasing in size. If probe level interactions are evaluated, the analysis space is of dimensionality for probes on the genome. As a result, the analysis time and memory usage will also increase quadratically with the number of probes. Instead of a grid positioned at the genomic positions of the probes, we employ an equally spaced genomic grid as a basis for all subsequent steps. The distance between grid-points is a user-defined variable, and will determine the finest resolution of the outcome and computational efficiency. We have performed all analyses using a genomic grid with a grid spacing of 20 Kb. Given a genome of base pairs and a grid spacing of, this results in grid positions, with, where represents the integer part of the real number,. The grid positions can be represented in the following row vector:, where. Let the aCGH profile of the tumor be represented by the following row vector of probe measurements:, with being the number probes. Let the midpositions of the probes be located at. To employ the genomic grid we need to compute, for each aCGH array, the value of the aCGH profile on the grid points. We achieve this by performing, for the grid position,, a kernel-weighted regression of all probe values situated in the range, employing a triangular kernel centered at, with maximal amplitude of 1 and width of 2. More specifically, the interpolated copy number aberration at the grid position is given by: (1) Here the set is the set of probe positions such that. The interpolated copy number profile of the tumor is represented by the row vector:. The complete dataset of tumors is refopresented by the matrix, where the probe values of the tumor constitute the row of matrix. Negative and positive log2 values respectively denote loss or gain of DNA in the test sample versus the reference sample. We regard both situations separately, which prevents the negative and positive values cancelling each other through summation later in the algorithm. We separate gains and losses by only retaining grid positions with positive values for the gains or negative values for the losses. The absolute values of the separated matrices are then used in the downstream steps. The remaining grid positions are set to zero. More specifically, the gains matrix, is given by, with (2) Similarly, the loss matrix, is given by with (3) Because we treat gains and losses separately we have four different co-occurrence situations to be considered given two loci on the genomic grid: i) gain/gain, ii) gain/loss, iii) loss/loss and iv) loss/gain. So, when evaluating the co-occurrence of loci and, we will evaluate the behavior of i) columns and for gain/gain; ii) columns and for gain/loss; iii) columns and for loss/loss and iv) columns and for loss/gain. (Here is the column of matrix). All subsequently described steps will be performed for these four situations separately, where and will be employed as shorthand for the abovementioned column vectors of interpolated copy number values associated with genomic grid positions and, respectively. The first component of the co-occurrence score is the continuous variant of the AND Boolean logic function: the minimum operation. For two grid points, and, the sum across all tumors of the minimal probe value per tumor at and,, is calculated as follows: (4) These values are aggregated in a matrix,. If we only use the minimum as a scoring function, those grid positions that are ubiquitously aberrated will always receive a high score, regardless of the aberration pattern in the other grid position. Two regions that are aberrated ubiquitously in all tumors are undoubtly important to the tumor but they are not necessarily functionally related. They might be a hallmark of the particular disease under study, but show no direct functional interaction. To prevent these ubiquitously aberrated regions from dominating the analysis and to detect those regions that represent functional co-occurrences, we weigh the minimum score computed above with the covariance of the interpolated probe values at the two grid positions and, (5) where and are the expected values of the probe values at grid position and across tumors, respectively (i. e. and). These values are aggregated in a matrix,. We combine both the minimum matrix and the co-variance matrix by element-wise multiplication to form the co-occurrence score matrix,, with (6) Since we believe the co-occurrence score to be a smooth variable, and since neighboring co-occurrence values can therefore be employed to reduce the noise locally, we convolve the co-occurrence score matrix with an isotropic 2D Gaussian kernel function. In practice this implies sampling the 2D Gaussian kernel function on a square grid consisting of × genomic grid positions and then performing the convolution of this sampled kernel function with the co-occurrence score matrix. The sampled isotropic 2D Gaussian function is defined as, with (7) The standard deviation of the isotropic Gaussian,, determines the scale of the analysis. Since the Gaussian quickly decays we set, allowing contributions from, convolving with a finite kernel with minimal loss in accuracy. The scale of an analysis is therefore defined as. The scales employed in this study are: 2 Mb (= 1/3 Mb), 10Mb (= 5/3 Mb) and 20Mb (= 10/3 Mb). Before the convolution step, we pad the co-occurrence matrix by mirroring the true data at each chromosome boundary and each centromere. By convolving the appropriately padded co-occurrence score matrix and the sampled 2D Gaussian function the Convolved Co-occurrence Matrix (CCM) is obtained: (8) with, as the convolution operator and (9) This matrix is a representation of the amount of co-occurrence between two locations on the genome. We calculate a CCM-matrix for each possible combination of chromosome-arms and for each of the four combinations of gains and losses listed above. With 39 unique chromosome arms in the human genome (disregarding the p-arms of the acrocentric chromosomes and the sex chromosomes), three different scales and 4 triangular pair-wise matrices to evaluate (loss-loss, gain-gain, gain-loss and loss-gain) we compute 8892 different CCMs. To solve this problem computationally we used a large distributed computing cluster. Our choice of resolution of the genomic grid was bounded by the memory present on the nodes. We set to 20000 base pairs, which is the lowest value still allowing the largest chromosome-arm pair to be successfully computed on one computing node. For each CCM we determine the top N peaks for each combination of gains and losses. The nth peak represents two co-occurring loci, and, and the location of the peak is defined by two co-ordinates on the genomic grid:. For each locus, we define a region of interest of size centered on and, respectively. We define this small region of interest to only select regions that are very near to the actual peak. To investigate the co-occurrence for functional relationships, we extract, for each of the co-occurring loci, the genes present in the regions of interest. More specifically, we define, for loci and, the associated gene sets and, where (10) and (11) Where is the position of gene, which we chose to be the mid-position of the gene. The genesets were established by a BioMart query from the Ensembl database. We restricted ourselves to the bio type ‘protein_coding’. The list of CGC genes was obtained from the CGC website (http: //www. sanger. ac. uk/genetics/CGP/Census/). The reference list of all genes was retrieved from the Ensembl website, with a filter to keep only genes with bio-type = ‘protein_coding’. This left 18840 genes. All CGC genes that could not be mapped back to the reference gene set were excluded. The CGC genes that were annotated as ‘recessive’ were used as the tumor-suppressor genes and ‘dominant’ as oncogenes. Enrichment for all CGC genes, the tumor-suppressor subgroup and the oncogene subgroup in the gene sets determined by the co-occurrence analysis was calculated using a Fisher' s exact test. The set of pairs of interacting genes which are such that one gene is associated with locus and the other gene of the pair with locus is then defined as (12) Where represents the confidence of interaction, according to the STRING database, between genes gk and gl. We then determine all gene lists of interactors for the top N peaks of a given co-occurrence analysis, i. e. : (13) For each of the top N co-occurring loci, we also determine the total number of genes in the regions of interest of those loci. So, for loci and we define the set: (14) The total number of genes associated with the top N co-occurring loci is then given by (15) The interaction ratio,, is then defined as (16) where denotes the cardinality of set. As a control we randomly pick size-matched locations for all co-occurring regions in the top N and repeat the process for recovering interactions. For 100 randomly chosen co-occurring regions we calculate the resulting. A Fisher' s exact test is then used to asses the significance of enrichment of versus. For all pairs of co-occurring loci,, present in the top N of an analysis, let the set of loci representing the first and second member of the co-occurrence locus be defined asandrespectively. Given that the pairs of genomic locations corresponding to the top N co-occurring loci are given bywe define the set of genomic locations loci involved in co-occurrences asFor each possible pair of locations in the genomic distance is aggregated in matrix: (18) Where is defined as: (19) We perform hierarchical clustering on matrix using single linkage hierarchical clustering. Leaf nodes are assigned to clusters using a distance cutoff of 107 bp (10Mb). Clusters are represented as nodes in a graph. Edges between nodes are drawn if any co-occurrence relationship is found between loci present in the nodes. The case we subjected to analysis was a dataset containing 105 cell-lines derived from hematological origin. The aCGH measurements were done on 1. 8 million probe Affymetrix SNP 6. 0 arrays. After data pre-processing we were left with 95 samples. These cell lines are a subset of the Cancer Genome Project cancer cell line project (http: //www. sanger. ac. uk/genetics/CGP/CellLines/). A list of the cell lines included in this dataset can be found in Dataset S1. | Title: Identification of Networks of Co-Occurring, Tumor-Related DNA Copy Number Changes Using a Genome-Wide Scoring Approach Summary: It is generally accepted that a normal cell has to acquire multiple mutations in order to become a malignant tumor cell. Considerable effort has been invested in finding single genes involved in tumor initiation and progression, but relatively little is known about the constellations of cancer genes that effectively collaborate in oncogenesis. In this study we focus on the identification of co-occurring DNA copy number alterations (i. e., gains and losses of pieces of DNA) in a series of tumor samples. We describe an analysis method to identify DNA copy number mutations that specifically occur together by examining every possible pair of positions on the genome. We analyze a dataset of hematopoietic tumor cell lines, in which we define a network of specific DNA copy number mutations. The regions in this network contain several well-studied cancer related genes. Upon further investigation we find that the regions of DNA copy number alteration also contain large networks of functionally related genes that have not previously been linked to cancer formation. This might illuminate a novel role for these recurrent DNA copy number mutations in hematopoietic malignancies. | 10,038 | 265 | lay_plos | en |
Summarize: Sub-title: Trend "has flipped" says researcher Picture: It used to be that the kids out in the bushes smoking marijuana were the trouble makers, but new research shows that this group academically outperforms their peers who smoke both marijuana and tobacco. In one of the largest, long-term studies of substance co-use among teens, Dalla Lana School of Public Health researchers examined trends of tobacco and marijuana use from 1981 to 2011 and found that marijuana smokers receive higher grades than those who smoke both substances. “In the past, cannabis use was associated with more problematic behaviours, but this trend has flipped,” said Michael Chaiton, assistant professor in epidemiology and public health policy at U of T’s Dalla Lana School of Public Health. “Youth tobacco users are likely to have poor academic performance and engage in socially deviant behaviours, like vandalism, theft or assault.” The study, published in the Journal of School Health’s March issue, analyzed self-report survey data from the Centre for Addiction and Mental Health’s Ontario Student Drug Use and Health Survey, including a total of 38,331 students in grades 7, 9 and 11. A user and/or co-user are defined as someone reporting daily tobacco and/or marijuana use in the past month. Poor academic performance, which is linked to increased risk of tobacco, marijuana and a variety of other risky behaviours, was measured as an indicator of problem behaviour to see if it was related to substance use or co-use. Marijuana use peaked about a decade ago, but overall attitudes towards the drug have become more normalized, so many teens see it as safer than tobacco, although this is not inherently true, researchers say. Smoking rates have declined by about six per cent among school-aged youth in the last decade, but researchers believe smoking cigarettes has become a new signal of social deviation in this group. “Youth smokers are becoming a more vulnerable population with high levels of substance use and mental health comorbidities,” said Chaiton, who is also a scientist at the Ontario Tobacco Research Unit. Other interesting study insights include: 92 per cent of teen cigarette smokers also use marijuana; 25 per cent of teen marijuana users also smoke cigarettes; In 1999, co-use was at an all-time high at 12 per cent, with boys in lower grades most likely to be a part of this group; Students with lower academic performance were more likely to be co-users than users of either substance alone; There’s a growing “straight edge” cohort, with 90 per cent of Ontario students not using either substance. “Drug prevention programs should be aligned with student realities, which means acknowledging and addressing patterns of co-use,” said Maritt Kirst, co-author of the study and assistant professor in social and behavioural health sciences at U of T’s Dalla Lana School of Public Health. “This study identifies which youth are most at risk and can help public health professionals tailor prevention programs accordingly.” Study authors also suggest that marijuana prevention programs may take a renewed focus on the drug’s harmful effects instead of underscoring the drug’s illegal status – the traditional approach of such programs. Nicole Bodnar is a writer with the Dalla Lana School of Public Health at the University of Toronto. Andrea Janus, CTVNews.ca Students who only smoke marijuana do better at school than classmates who smoke just tobacco, or who smoke both tobacco and pot, says a new study, which tracked substance use among teens over 30 years. Researchers from the University of Toronto’s Dalla Lana School of Public Health analyzed data from a survey administered to nearly 39,000 Ontario students between 1981 and 2011. The Centre for Addiction and Mental Health asked students in Grades 7, 9 and 11 about their tobacco and marijuana use, and their academic performance. The study found that marijuana-only users did better at school than their counterparts who smoked only cigarettes or who smoked both cigarettes and marijuana. However, the findings reflect the fact that fewer students smoke tobacco today compared to 30 years ago, and those that do make up a very “marginalized, vulnerable” population, says lead study author Michael Chaiton, assistant professor in epidemiology and public health policy. About 92 per cent of tobacco users also use marijuana, the study found. However, only 25 per cent of marijuana uses also smoke tobacco. “It’s better relatively,” Chaiton says of marijuana-only users’ academic performance. Marijuana users don’t outperform non-users, Chaiton says. “Now there is a distinction between marijuana use and co-use with other substances, and it’s an indication of the changing social norms. So it’s not an absolute that they do better; it’s that social norms have changed and the population of people who use marijuana are more like the general population.” The study was published in the March edition of the Journal of School Health. In the 1980s, when the study began, there was less marijuana use among students. And those who did smoke pot also smoked tobacco. At the time, pot use among tobacco smokers was very low. Thirty years later, that had switched, the researchers found. As tobacco use declined, marijuana use shot up. And among the remaining tobacco users, marijuana use is now very high. One reason for the statistical switch, Chaiton says, is the effectiveness of anti-tobacco messaging in recent years. “The population of youth smokers right now is one that is a fairly marginalized population, quite a vulnerable population, so they are at high rates of cannabis use but also of other drugs and other behaviours,” Chaiton says. “So the change in trends is that this is a social phenomenon. This is not that tobacco is causing this, it is something that has changed socially in the role of tobacco in society.” Now that marijuana smoking has become more of a social norm, Chaiton says, programs aimed at keeping youth from risky behaviours such as drug abuse must take into account two factors: that more students now smoke marijuana compared to 30 years ago, and that students who smoke tobacco are more likely to use marijuana or other drugs and engage in at-risk behaviours such as vandalism and theft. As marijuana use becomes more prevalent and socially acceptable, Chaiton says, the focus must turn to developing programs for youth that properly educate them on the risks. Tobacco and marijuana are “similar drugs in many different ways,” Chaiton notes, and “people dramatically underestimate the risks associated with cannabis use, particularly among youth.” “I would argue that we need to start talking about them in the same way and start addressing them in the same types of interventions,” he says, particularly given the growing public discussions about decriminalizing or legalizing marijuana. “If we do legalize or change the regulations in dramatic ways, that does change the social environment again and that can, as we’ve seen a number of times, cause big shifts in youth and we could see another big shift in marijuana use among youth.” | Summary: A little more ammo for the pro-marijuana crowd: A major study out of Canada finds that students who smoke pot do better in school than those who smoke tobacco or indulge in both, reports CTV. But, no, it's not about marijuana making kids smarter. In fact, kids who abstain from smoking of any kind do best of all. Chalk it up instead to the growing acceptance of pot. "It's an indication of the changing social norms," says the lead author of the study from the University of Toronto's Dalla Lana School of Public Health. "So it's not an absolute that they do better; it's that social norms have changed, and the population of people who use marijuana are more like the general population." The study looked at data over the last 30 years and compared kids in grades 7, 9, and 11 in 1981 with their counterparts in 2011. As the U of T News puts it, "it used to be that the kids out in the bushes smoking marijuana were the trouble makers," but the trend seems to have reversed itself. Now the kids smoking tobacco exclusively are the "marginalized" ones. The study found that 92% of students who smoke cigarettes also smoke pot, but only 25% of pot smokers also smoke tobacco. The biggest group was the 90% who reported smoking neither, and researchers say that's the way to go. "People dramatically underestimate the risks associated with cannabis use, particularly among youth," says the UT scientist. | 1,573 | 342 | multi_news | en |
Write a title and summarize: Spike-timing dependent plasticity (STDP) is a widespread plasticity mechanism in the nervous system. The simplest description of STDP only takes into account pairs of pre- and postsynaptic spikes, with potentiation of the synapse when a presynaptic spike precedes a postsynaptic spike and depression otherwise. In light of experiments that explored a variety of spike patterns, the pair-based STDP model has been augmented to account for multiple pre- and postsynaptic spike interactions. As a result, a number of different “multi-spike” STDP models have been proposed based on different experimental observations. The behavior of these models at the population level is crucial for understanding mechanisms of learning and memory. The challenging balance between the stability of a population of synapses and their competitive modification is well studied for pair-based models, but it has not yet been fully analyzed for multi-spike models. Here, we address this issue through numerical simulations of an integrate-and-fire model neuron with excitatory synapses subject to STDP described by three different proposed multi-spike models. We also analytically calculate average synaptic changes and fluctuations about these averages. Our results indicate that the different multi-spike models behave quite differently at the population level. Although each model can produce synaptic competition in certain parameter regions, none of them induces synaptic competition with its originally fitted parameters. The dichotomy between synaptic stability and Hebbian competition, which is well characterized for pair-based STDP models, persists in multi-spike models. However, anti-Hebbian competition can coexist with synaptic stability in some models. We propose that the collective behavior of synaptic plasticity models at the population level should be used as an additional guideline in applying phenomenological models based on observations of single synapses. Spike-timing dependent plasticity (STDP) is a form of activity-dependent synaptic plasticity that appears throughout the nervous system [1,2, 3]. In STDP, pairs of pre- and postsynaptic action potentials potentiate a synapse when the presynaptic spike precedes the postsynaptic spike, and depress it for the reverse order [4,5]. However, when multiple pre- and postsynaptic spikes occur across a synapse over a short interval of time, the resulting plasticity depends on their timing in a more complex manner. For example, pair-based STDP models predict that “pre-post-pre” and “post-pre-post” triplets of spikes with the same pairwise intervals should induce the same plasticity, but experiments indicate that these two triplet patterns have different effects [6,7]. This and similar contradictions motivated the development of multi-spike models of STDP, which go beyond pairwise interactions of pre- and postsynaptic spikes (see [8] for a review). Here, we analyze three such models that are based on experimental results to determine how they affect populations of synapses converging onto a postsynaptic neuron. We focus on two basic features: stability and competition. Stability is a property of the distribution of synaptic weights arising from an STDP model, and we will distinguish three cases: unstable, partially stable, and stable. In the unstable case, synaptic weights perpetually increase under STDP, unless some upper limit is imposed (in principle, weights could also decrease perpetually, but this is atypical). Although we briefly consider soft bounds to limit the range of synaptic weights, we primarily consider hard bounds. When hard limits are imposed on an unstable STDP model, the synaptic weights cluster tightly against the upper bound. Another more interesting case is partial stability, in which individual synaptic weights increase or decrease indefinitely, but the average of the weights across a synaptic population stays fixed. When hard bounds are imposed on a partially stable STDP model to limit the increases and decreases of individual synapses, the synaptic weights tend to cluster at either end of their allowed range, forming a U-shaped distribution [9]. Finally, when an STDP model is stable, no hard bounds need to be imposed, and synaptic weights form a unimodel distribution [10,11,12,13]. We are interested in determining whether, and under what parameter values, different multi-spike STDP models lead to stable, partially stable or unstable synaptic weight distributions. The impact of STDP on the weights of synapses onto a postsynaptic neuron depends on correlations between their presynaptic spike patterns. This can be studied by dividing the inputs to a neuron into two groups, one with correlated presynaptic activity and the other with uncorrelated presynaptic spiking. In this context, competition refers to the propensity of either a correlated or an uncorrelated group of synapses to gain control of the postsynaptic spiking, while the other group become less influential, both as a consequence of STDP. In cases that we call “Hebbian”, the synapses with correlated input become stronger than those with uncorrelated input. In other “anti-Hebbian” cases, the reverse occurs and the correlated synapses become weaker than the uncorrelated. We are interested in whether synaptic plasticity is Hebbian or anti-Hebbian, by this definition, in various multi-spike STDP models and for different parameter values of those models. The three multi-spike STDP models that we consider were proposed on the basis of different experimental results. In the “suppression model”, inspired by experimental results in cortical slices, the plasticity-inducing effect of each pre- or postsynaptic spike is suppressed by preceding spikes [6,14]. The “triplet model”, inspired by experiments in hippocampal slices, includes the effect of neighboring pre-post pairings as well as depression exerted by preceding presynaptic spikes and potentiation by preceding postsynaptic spikes [15]. The third model we consider, the “NMDAR-based model”, is phenomenologically based on the kinetics of the N-Methyl-D-Aspartate receptor [16]. This model was proposed before experimental results on multi-spike effects in STDP were available, and hence it was not explicitly aimed at accounting for multi-spike interactions, unlike the first two models. However, as we show in the Results section, it demonstrates a rich repertoire of multi-spike interactions, and can behave similar to either of the first two models depending on its parameters. Therefore, we feel it deserves to be considered as a multi-spike STDP model, even though it may not have been intended as such initially. We begin by reviewing results for pair-based STDP to establish our approach and introduce ways of characterizing the effects of plasticity. We then apply this approach and these characterizations to the multi-spike STDP models. For each case, we first consider the parameters originally proposed for the model, and then systematically explore a range of parameter values to evaluate stability and competition. In light of the results obtained, we conclude by discussing relationships between the models at the biophysical level, and the computational implications of each model at the synaptic population level. To explain our method for analyzing synaptic stability and competition and also to provide a benchmark of comparison for the multi-spike STDP models, we first examine a pair-based STDP model. In this model, synapses are modified only on the basis of the intervals between pairs of pre- and postsynaptic spikes. When a synapse receives a larger ensemble of spikes, such as triplets or quadruplets, plasticity is induced by the pre-post pairs within the ensemble independent of the higher-order structure of the ensemble. As stated in the introduction, similarly spaced “pre-post-pre” and “post-pre-post” triplets induce the same amount of synaptic modification in this model (Fig 1A and 1E). The parameters of the pair-based model include the maximum amounts by which synapses can be potentiated or depressed, A+ and A−, and the time constants for the potentiation and depression windows, τ+ and τ− (Eq 10). These parameters also appear in the multi-spike models. To quantify the average modification of a synapse under STDP, we first calculate the probability of a pairing interval Δt for spikes arriving at the synapse and then average synaptic modification (Eq 10) over that probability. We assume that the pre- and postsynaptic spike trains are both Poisson. The rate of the presynaptic spike train takes the constant value rpre. The baseline rate of postsynaptic firing is denoted by rpost (Eq 8). When a postsynaptic action potential is generated, presynaptic spikes are equally likely to arrive at any later time because the postsynaptic spike has no effect on presynaptic activity. However, when a presynaptic spike arrives at a particular synapse, it transiently increases the postsynaptic firing rate by an amount proportional to the strength of that synapse (Eq 9). As a result, a postsynaptic action potential is more likely to be induced shortly after the arrival of a presynaptic spike. Including both the baseline rate and this brief enhancement, the average synaptic modification or the “drift” for a synaptic strength w is (see Methods) dwdt= (A+τ+−A−τ−) rprer¯post+A+τ+τsrprew (τs+τ+) (Vth−Vr) τm. (1) The first term in this equation relates the change in synaptic strength of a particular synapse, w, to the average strength of all the excitatory synapses, ⟨w⟩, through the dependence of the baseline firing rate r¯post on this average. This term is the same for all synapses, so we call it the “baseline drift”. The second term depends on the synaptic strength of the particular synapse being considered, and it arises from the transient increase of postsynaptic firing rate following a presynaptic spike at this synapse. We call it the “w-dependent drift”. The rate of change of the average of all the excitatory synaptic weights is given by the sum of the baseline drift and the average of the w-dependent drift, d⟨w⟩dt= (A+τ+−A−τ−) rprer¯post+A+τ+τsrpre⟨w⟩ (τs+τ+) (Vth−Vr) τm. (2) The average synaptic strength in the steady-state is the values of ⟨w⟩ that sets the right side of Eq (2) to zero (i. e. a fixed point). One such fixed point occurs when all the synapses are zero (⟨w⟩ = 0). This makes the postsynaptic neuron silent (r¯post=0) and sets both the baseline and average w-dependent drifts to zero. This state is uninteresting and simply reflects the fact that no plasticity occurs when the postsynaptic neuron is silent. If the synaptic strengths are not zero, the average w-dependent drift is always positive because presynaptic spikes always enhance postsynaptic firing. As a result, a nontrivial fixed point for the average synaptic weight can occur only if the baseline drift is negative (A− τ− > A+ τ+) so that it can cancel the w-dependent drift (Fig 1B, closed circle). This fixed point is stable, because the positive w-dependent drift dominates if the average weight is smaller that the fixed-point value, and the negative baseline drift dominates if it is larger. Mathematically, stability requires the slope of the average drift to be negative at the fixed point (Fig 1B), which always holds for the nontrivial fixed point of the pair-based model. In summary, the steady-state average synaptic strength in pair-based STDP has a stable nontrivial mean if the depression window is larger than the potentiation window (A− τ− > A+ τ+). This fixed point is unique, so the mean of the steady-state distribution of synaptic weights converges to this value regardless of its initial value. The stability of the mean is not a sufficient condition for the steady-state distribution of synaptic strengths to be fully stable, each synapse must also have a stable deviation from the mean. The strength of a particular synapse can be expressed as w = ⟨w⟩ + δw, where δw is the deviation of the synapse from the mean. If the deviation tends to grow over time, the synapses will drift away from the mean and the distribution will be partially stable and U-shaped (bimodal). If the deviation tends to decrease, the synapses will cluster around the mean and the distribution will be stable and unimodal. Assuming that the mean synaptic strength is at steady-state and that the deviation of an individual synapse (out of a few thousand) does not alter the mean significantly, the change of the deviation over time is governed solely by the w-dependent drift and can be derived from Eq (1) as dδwdt=A+τ+τsrpreδw (τs+τ+) (Vth−Vr) τm. (3) Because the coefficient of δw in this equation is positive (Fig 1B, inset), the deviations tends to grow, and the final distribution of synaptic strengths for pair-based STDP is partially stable and U-shaped even though the mean is stable (Fig 1C, ref. [9]). To check the accuracy of Eqs (2) and (3), we computed the synaptic drift by averaging the amount of induced synaptic modification in simulations lasting 03 s of simulated time, without implementing synaptic modification (Fig 1B, gray shade). In general, the agreement is good; the discrepancy between the analytic and simulation results at low average synaptic strengths is due to the fact that our approximation for the transient postsynaptic firing rate (Eq 9) is only accurate when the mean excitatory input is significantly larger than the mean inhibitory input. In the parameter regime where the potentiation window is larger than the depression window (Fig 1E–1H), the mean synaptic weight only has the trivial and unstable zero fixed point (Fig 1F), so the distribution is unstable and all of the synaptic strengths grow until they hit the upper bound, regardless of their initial values (Fig 1G). When the mean synaptic strength is stable and the w-dependent drift is positive, it is possible for STDP to discriminate between two groups of synapses based on the degree of correlation in their presynaptic spike trains. If the spike trains arriving at one group of synapses are correlated and those of the other synapses are not, the correlated group induces a larger transient increase in the postsynaptic firing rate and hence a larger w-dependent drift. Therefore the correlated group is more likely to become stronger than the mean, and the uncorrelated group tends to become weaker to maintain the balance around the mean (Fig 1D). This results in a Hebbian competition among the synapses [9]. On the other hand, when there is no stable mean, all the synapses tend to grow regardless of their correlation and no competition takes place, although the correlated synapses still end up stronger than the uncorrelated group (Fig 1H). Therefore, the condition for Hebbian competition through pair-based STDP is the existence of a stable mean, i. e. A− τ− > A+ τ+, which is equivalent to partial stability. Importantly, as long as the steady-state mean of the synaptic strength is within the allowed range, this condition is not changed by modifying the lower or upper bounds of the synaptic strengths. This is also the case for the multi-spike models discussed in the following sections. Experimental results on synapses in hippocampal cultures reveal a marked asymmetry in the plasticity induced by post-pre-post and pre-post-pre spike sequences, in contrast to the predictions of the pair-based model (Fig 1A and 1E). Post-pre-post sequences induce potentiation, and pre-post-pre has little or no effect [7]. In addition, in experiments on cortical synapses, the balance between potentiation and depression shifts toward potentiation when the frequency of pre-post pairing events increases, another property not captured by pair-based STDP [17]. These results motivated Pfister & Gerstner [15] to propose the triplet model, which takes into account interactions of spikes beyond pre-post pairings. In addition to the effect of pre-post pairings, the triplet model includes additional depression due to previous presynaptic spikes and additional potentiation from earlier postsynaptic spikes (Fig 2A). This is accomplished through a presynaptic depression variable and a postsynaptic potentiation variable assigned to each synapse (Eq 11). In the absence of incoming presynaptic spikes, the presynaptic depression variable decays exponentially with time constant τpre. Likewise, the value of postsynaptic potentiation variable decreases exponentially in the absence of postsynaptic spikes with time constant τpost. When a presynaptic spike reaches the synapse, the presynaptic depression variable abruptly increases by the amount Apre, and when a postsynaptic spike occurs, the postsynaptic variable increases by Apost (Eq 12). This is how the triplet model accounts for the asymmetry of synaptic modification in response to triplets. For a pre-post-pre triplet, the first presynaptic spike induces extra depression on the synapse, while for a post-pre-post triplet the first postsynaptic spike induces extra potentiation (Fig 2B). The triplet model that we consider sums the contributions of all previous pre- and postsynaptic spikes as well as all pre-post pairings (all-to-all). Pfister & Gerstner [15] also provided a version of the triplet model based only on nearest neighboring spikes, but the qualitative behavior of both versions is similar. As we did for pair-based STDP, we can derive equations governing the evolution of the mean synaptic strengths and deviations around the mean for individual synaptic weights. The average values of the presynaptic depression and postsynaptic potentiation variables, obtained from substituting rates for spikes in Eq 12, are Apre τpre rpre and Apostτpostr¯post. Using these values and averaging the synaptic modification (Eq 11) over the probability of pre-post pairings, the drift of the mean of the synaptic weights in the triplet model is d⟨w⟩dt=A+τ+rprer¯post+Apostτpostτ+rprer¯post2−A−τ−rprer¯post−Apreτpreτ−rpre2r¯post+ (A++Apostτpostr¯post) τ+τsrpre⟨w⟩ (τs+τ+) (Vth−Vr) τm. (4) As in the pair-based model, the last term in this equation is the w-dependent drift and the other terms make up the baseline drift. The dynamics of deviations of individual synapses from the mean is governed by the w-dependent drift, so dδwdt= (A++Apostτpostr¯post) τ+τsrpreδw (τs+τ+) (Vth−Vr) τm. (5) As in the pair-based model, the coefficient of δw is always positive, so individual weights will drift away from the mean for any choice of parameters, making individual synaptic weights unstable. The parameters of the original model were fit by Pfister & Gerstner [15] separately to match experimental data from hippocampal cultures and cortical slices, resulting in two sets of parameters. Our simulation results indicate that, for both sets of parameters, the distribution of synaptic weights is unstable, so that all the synaptic weights cluster around the upper bound. In addition, no competition takes place between correlated and uncorrelated synapses with these parameter sets. This led us to consider properties of the triplet model for a range of parameter values. As in our discussion of pair-based STDP, we study the triplet model when pair-based potentiation is larger than pair-based depression (A− = 0. 005 mV, A+ = 1. 01A−) and when pair-based depression is larger than pair-based potentiation (A+ = 0. 005 mV, A− = 1. 01A+). In each case, we varied the ratio between postsynaptic potentiation and presynaptic depression (Apost/Apre) systematically, while keeping Apre constant at 0. 001 mV. We first examine the fixed points of the mean synaptic weight (Fig 3A and 3F). When Apost/Apre is small, the average synaptic weight has two nontrivial fixed points (Fig 3B and 3G). The first is stable (Fig 3B and 3G, filled circle) and the second is unstable (Fig 3B and 3G, open circle). The appearance of the unstable fixed point in the triplet model is due to the dependence of the postsynaptic potentiation on the postsynaptic firing rate. This added potentiation increases when the mean synaptic weight increases, eventually overcoming the combined effect of presynaptic and pair-based depression. The existence of two fixed points makes the steady-state distribution of synaptic weights sensitive to the initial distribution. If the mean of the initial distribution is greater than the unstable fixed point, the distribution will be unstable and all of the weights will be pushed toward the upper bound (Fig 3D and 3I, right). If the mean of the initial distribution is lower than the unstable fixed point, the mean of the steady-state distribution converges to the stable fixed point and individual weights drift away from the mean toward the lower and upper bounds, resulting in partial stability and a U-shaped distribution similar to the pair-based model (Figs 3D and 2I, left). When Apost/Apre reaches a critical value, the two fixed points coalesce and annihilate each other, and only the trivial unstable fixed point remains (Fig 3C and 3H). In this case, regardless of the initial distribution, the final distribution is unstable and tightly clustered near the upper bound (Fig 3D and 3I, bottom). As we argued in the case of the pair-based model, synaptic competition can only take place when the steady-state distribution has a nontrivial stable mean and is partially stable. In the triplet model, when Apost/Apre is relatively small and the initial mean synaptic weight is lower than the unstable fixed point, this condition is fulfilled (Fig 3A and 3F, dark gray areas; Fig 3E, left). However, if the stable and unstable fixed points are too close together, there is no guarantee of synaptic competition (Fig 3J, left). The reason for this is that when a subset of the synaptic inputs are correlated, presynaptic spikes tend to arrive in tandem and induce large transients in the postsynaptic firing rate, causing large fluctuations in the mean synaptic weight. This can cause the mean synaptic strength to fluctuate beyond the unstable fixed point, destabilizing the weight distribution. As a result, the parameter regime for synaptic competition in the triplet model is highly restricted to the region of small Apost/Apre with A+ < A− (Fig 3A, dark gray). Even within this small region, if the correlation coefficient among the correlated synapses is high, competition does not take place and all the synapses tend to the upper bound (S1 Fig), due to the fluctuations mentioned above. Thus, it is not surprising that the original parameters obtained by Pfister & Gerstner [15] did not lead to competitive synaptic plasticity. In summary, the novel properties of the triplet model, as compared to the pair-based model, are the sensitivity to the initial distribution of weights and a tighter parameter range for Hebbian competition. Plasticity experiments in cortical slices using triplets of spikes showed different effects than the hippocampal results. In the synapses of the visual cortex of rats, pre-post-pre triplets induce potentiation whereas post-pre-post triplets induce depression [6]. These results led Froemke et al. [6] to propose the suppression model, in which plasticity is induce by nearest neighbor pre- and postsynaptic spikes. The plasticity is computed from the standard pair-based STDP curve, but the effect of the presynaptic spike in each pair is suppressed by previous presynaptic spikes and, similarly, the plasticity induced by the postsynaptic spike in each pair is suppressed by previous postsynaptic spikes (Fig 4A). The suppression is maximal immediately after each pre- or postsynaptic spike, and it decreases exponentially as the interval between consecutive pre- or postsynaptic spike increases (Eq 13). The suppression accounts for the asymmetry of synaptic modification in response to triplets. In the case of a pre-post-pre triplet, the first pair (pre-post) induces potentiation, but the amount of depression induced by the second pair (post-pre) is suppressed by the first presynaptic spike. For a post-pre-post triplet, the first pair (post-pre) induces depression, but the potentiation induced by the second pair (pre-post) is suppressed by the first postsynaptic spike (Fig 4B). The parameters of the model were originally set to match the synaptic modification seen in the experiments (ref. [6]; Table 2). Our numerical simulations with these parameters show that the steady-state distribution is unstable and tightly clustered around the upper bound. When correlations are induced in half of the synaptic inputs, no competition takes place and all the weights are potentiated indiscriminately. To observe a range of behaviors of this model, we set the suppression time constants equal to the values given by Froemke et al. [6], namely τpre = 28 ms, τpost = 88 ms. We also set the maximum potentiation and depression values equal (A+ = A− = 0. 005 mV and fixed the depression time constant (τ− = 20 ms). We then varied the potentiation time constant τ+ to observe different behaviors of the model. Transitions to different behaviors can also be seen when changing other parameters (for example the ratio A+/A−), but our simulations showed that changing the ratio between the potentiation and depression time constants (τ+/τ−) reveals these transitions most clearly. Calculating the drift of synapses in the suppression model is more complicated than in the models considered above. We leave the details to S2 Appendix and report the results here. When τ+/τ− < 1. 2, the average synaptic weight has a stable nontrivial fixed point (Fig 5A–5C). For higher values of τ+/τ−, the nontrivial fixed point disappears and the average synaptic weigh has only the trivial zero fixed point (Fig 5A and 5D). For low τ+/τ− values, the steady-state distribution of weights is partially stable and U-shaped, as in the case of the pair-based model (Fig 5E). However, for τ+/τ− between 1. 05 and 1. 2, the value of the average synaptic weight grows rapidly (Fig 5A, gray area), and the steady-state distribution is stable and unimodal (Fig 5F), implying that the w-dependent drift is negative in this range. Because of the complexity of spike interaction in the suppression model, a complete characterization of the w-dependent drift is beyond our analytical calculations (S2 Appendix). However, features of the response of an integrate-and-fire neuron to a pair of presynaptic spikes in the context of the suppression model explain why the w-dependent drift becomes negative when the average synaptic weight is large. Suppose that two presynaptic spikes arrive at a neuron in quick succession, and we want to analyze the role of the second spike in inducing plasticity under the suppression model (Fig 6). The second presynaptic spike participates in plasticity twice: once by pairing with the previous postsynaptic spike, and again by pairing with the next postsynaptic spike. When the strength of the synapse is low, the first presynaptic spike is not very likely to induce a postsynaptic action potential after its arrival, so the pairing interval between the second presynaptic spike and the preceding postsynaptic spike is typically long, which induce weak depression (Fig 6A). However, if the synapse is strong, the first presynaptic spike is likely to induce a postsynaptic action potential, and its pairing interval with the second presynaptic spike is then short, inducing strong depression (Fig 6B). In addition, because of the high probability of postsynaptic firing in response to both presynaptic spikes, the interval between the induced postsynaptic spikes is short, which strongly suppresses the potentiation caused by pairing the second presynaptic spike with its following postsynaptic spike. Therefore, depression dominates over potentiation in the suppression model when synapses are strong. When this happens, deviations to even higher values lead to depression. This explains why w-dependent drift is negative when the average synaptic weight is large, which occurs when τ+/τ− approaches the critical value 1. 2 (Fig 5A, gray area). When half of the synapses receive correlated spike trains and the other half uncorrelated inputs, a distinctive features of the suppression model is that anti-Hebbian competition takes place: the uncorrelated synapses become strong and the correlated ones weak (Fig 5H–5J). This is the result of postsynaptic suppression. When correlated presynaptic spikes arrive, they tend to induce a postsynaptic spike shortly after their arrival. This makes the interval between the induced postsynaptic action potential and the previous spike shorter than for the postsynaptic response to uncorrelated input. As a result, potentiation is suppressed for correlated synapses, and they eventually lose the competition with uncorrelated ones. In analogy with what was described in the previous paragraph, correlated inputs are similar to inputs with strong synapses and, in either case, the high probability of postsynaptic spiking makes the w-dependent drift negative. In summary, the characteristic properties of suppression model are anti-Hebbian competition and stability of the synaptic distribution when the mean synaptic strength is large. The NMDAR-based model [16] was proposed as an explanation for the original STDP experiments of Markram et al. [4], and it predates both the triplet and suppression models and the data that inspired them. Nevertheless, as we will see below, it has features that resemble both of these models, and it is sensitive to spike interactions beyond pre-post pairings. The original version of the NMDAR-based model [16] includes the dynamics of the probability of presynaptic vesicle release. We focus on a simpler version that only models the modification of synaptic strengths by pre- and postsynaptic spikes [18]. In the NMDAR-based model, the NMDAR is assumed to have three states, rest, up and down. Each incoming presynaptic spike moves a portion of the NMDARs in the rest state into the up state, and each postsynaptic spike transitions a portion of the rest-state NMDARs into the down state. The NMDAR decays back to the rest state exponentially in the absence of spikes (E 14). In accord with the molecular kinetics of NMDARs [19,20], the rest state can be interpreted as an NMDAR that is not bound to glutamate and is blocked by Mg2+, the up state as an NMDAR that is bound to glutamate but blocked by Mg2+, and the down state as an NMDAR that is not bound to glutamate but has had its Mg2+ block removed by a postsynaptic spike. The model also has two second messengers, called “up” and “down” messengers, which mediate potentiation and depression, respectively. These can be in either active or inactive states. When a presynaptic spike arrives, a fraction of the inactive down messengers transition to the active state. Likewise, when a postsynaptic spike reaches the synapse, it moves a portion of the inactive up messengers into their active state. The messengers decay back to their inactive states in the absence of spikes (Eq 15). Finally, upon arrival of a presynaptic spike, the synapse is depressed proportional to the amount of active down messenger, provided that this is larger than a threshold θdn. Similarly, each postsynaptic spike causes the synapse to potentiate proportional to the amount of active up messenger provided that it is larger than a threshold θup (Eq 16). Thus, the presynaptic spike plays three roles in this model: it moves resting NMDARs into the up state, it activates the down messenger, and it induces depression. The postsynaptic spike also has three roles: it transitions resting NMDARs into the down state, activates the up messenger, and induces potentiation (Fig 7A). A key feature of the NMDAR based model is that preceding spikes decease the amount of available resting NMDARs available to upcoming spikes. This implements a mechanism akin to the suppression model, in which previous spikes suppress the effect of subsequent spikes. The roles of the second messengers are quite similar to those of the presynaptic depression and postsynaptic potentiation variables in the triplet model in that both integrate the effects of pre- and postsynaptic spiking to modify depression and potentiation. In fact, if we assume that the spikes have access to an unlimited pool of resting NMDARs and messengers, the NMDAR-based model is equivalent to the triplet model. Given the multi-spike interactions in the NMDAR-based model, it is not surprising that it responds asymmetrically to triplets of spikes. Our numerical simulations using the parameters provided by Senn et al. [16] (Table 2) show that the synaptic modification in response to triplets in this model is qualitatively similar to that of the suppression model (Fig 7B). The simulations also show that, with the parameters provided by Senn et al. [16], the steady-state distribution is unstable and tightly clustered around the upper bound. When correlations are induced in half of the synaptic inputs, no competition takes place and all the weights are potentiated indiscriminately. To examine the spectrum of behaviors in the NMDAR-based model, we calculated the synaptic drift (S3 Appendix). Interesting transitions into different regimes occur when the threshold of the up messenger is larger than that of the down messenger (θdn = 0. 2, θup = 0), and the ratio between maximum potentiation and maximum depression (A+/A−) is varied (Fig 8). All other parameters of the model are held constant at equal values for potentiation and depression components, and the time constants are set to the values provided by Senn et al. [16] (Table 2). When A+/A− is smaller than a critical value (0. 042), the average synaptic weight has both stable and unstable nontrivial fixed points. At the critical value, these two fixed points coalesce and disappear, and beyond the critical value the average synaptic weight has only the trivial fixed point at zero (Fig 8A). The sign of w-dependent drift also changes as A+/A− varies. When A+/A− is smaller than 0. 025, the w-dependent drift is negative, and for larger ratios it is positive (Fig 8B). Taken together, three different behaviors are observed in the NMDAR-based model: 1) When a stable mean synaptic weight exists and w-dependent drift is negative (0 < A+/A− < 0. 025, Fig 8A–8B, dark gray area), the steady-state distribution of synaptic weights is stable and unimodal (Fig 8C and 8F). 2) When a stable mean synaptic weight exists and w-dependent drift is positive (0 < A+/A− < 0. 042, Fig 8A and 8B, light gray area), the steady-state distribution of synaptic weights is partially stable and U-shaped (Fig 8D and 8G). 3) When the mean synaptic weight has no stable fixed point (A+/A− > 0. 042), the steady state distribution is unstable, and it clusters near the upper bound (Fig 8E and 8H). Synaptic competition is different in these three regions of the parameter space. When half of the input spike trains are correlated, the competition in the first region is anti-Hebbian because the w-dependent drift is negative and correlated synapses receive more depression (Fig 8I). It is Hebbian in the second region because the w-dependent drift is positive (Fig 8J). As in the triplet model, the closeness of the stable and unstable fixed points in this region makes synaptic competition elusive, such that when the correlation coefficient between the synapses is high, all the synapses tend to the upper bound and no competition takes place (S1 Fig). There is no competition in the third region because the mean is not stable (Fig 8K). In short, the distinguishing features of the NMDAR-based model compared to the pair-based model are the possibility of a stable synaptic distribution and anti-Hebbian competition when the maximum depression is significantly larger than the maximum potentiation. In previous sections, we imposed so-called hard bounds on the synaptic strengths to confine them between zero and a maximum allowed value (wmax). It is also possible to confine the synapse by implementing soft bounds, that is, by making the maximum depression and potentiation weight-dependent so that when a synaptic strength approaches the bounds, its rate of change gradually decreases. This can be done by multiplying A+ and A− by 1 − w/wmax and w/wmax respectively. In the case of the triplet model, the presynaptic depression and postsynaptic potentiation variables should be also multiplied by 1 − w/wmax and w/wmax respectively, because they appear as potentiation and depression factors as well. The steady-state distribution of synaptic strengths is stable and unimodal for all three multi-spike STDP models with soft bounds (Fig 9). This behavior is robust and holds for a wide range of parameters (we only show simulation results for the original parameters (Table 2) in each model). The soft bounds weaken synaptic competition drastically, so than the distributions of correlated and uncorrelated synapses are close to each other (Fig 9, insets). As has been shown for pair-based STDP [10,11], soft bounds turn STDP into a homeostatic plasticity mechanism with minimal sensitivity to the correlation structure of the external input. The main focus of this study has been on synaptic stability and competition, two desirable but often conflicting features of activity-dependent plasticity rules [21]. Our analytical tool for assessing these properties was calculating the drift of a population of synapses under each multi-spike STDP model. This method has been applied to the pair-based STDP model in a number of previous studies. The pair-based model with hard bounds was shown to produce a partially stable U-shaped steady-state distribution of weights and Hebbian competition that favors correlated synapses over uncorrelated ones [9,22,23]. On the other hand, the pair-base model with soft bounds has been shown to have a stable steady-state distribution at the expense of losing synaptic competition and sensitivity to input correlations [10,11]. Our analysis can be viewed as a reconfirmation of these results of the pair-based models and an extension into the domain of multi-spike STDP models. Our goal has not been to identify a superior model among the different options. Rather, we have highlighted the largely overlooked consequences of implementing these models at the population level. Table 3 summarizes the results of our survey of stability and competition in multi-spike STDP models. Like the pair-based model, the triplet model produces a partially stable steady-state distribution of synaptic weights and Hebbian synaptic competition. However, competition is observed only for a limited range of its parameters. The suppression model shows predominantly anti-Hebbian competition and a stable steady-state distribution of synaptic weights when the average weight is high. The NMDAR-based model displays both stable and partially stable steady-state distributions depending on the parameters, with anti-Hebbian competition in the former case and Hebbian in the latter. Our results indicate that the dichotomy between stability and Hebbian competition, which is well characterized for pair-based STDP models, persists in multi-spike STDP models. However, anti-Hebbian competition can coexist with full synaptic stability for at least some parameter regimes in the suppression and NMDAR-based models. Conflict exists between stability and Hebbian competition because, for such competition to take place, correlated synapses, which induce a large transient increase in postsynaptic firing rate, should be strengthened. This property undermines full stability of the synaptic distribution because it creates a positive feedback loop in which strong synapses, which also induce large transient increases in postsynaptic firing, become even stronger. Thus, it is not surprising that this conflict persists in more elaborate multi-spike models. Anti-Hebbian competition, on the other hand, involves weakening of correlated synapses that induce large transients in the postsynaptic activity, and so should be compatible with full stability of the synaptic distribution. The dichotomy between synaptic competition and stability is a specific form of the general stability/plasticity dilemma [24]. Every form of plasticity faces the challenge of maintaining the balance between forming new memories through modification of synaptic strengths, and preserving old synaptic configurations to maintain old memories. A more specific aspect of this challenge is that Hebbian (or anti-Hebbian) competition among synapses in a network is a powerful mechanism for shaping and modifying neural activity based on the properties of the inputs to the network. However, unless changes in synaptic strength are stabilized appropriately, the level of activity in a neural circuit can grow or shrink in an uncontrolled way [25]. Therefore, it is highly desirable, from a computational point of view, to find a biologically plausible model that reconciles synaptic stability with competition. A number of solutions have been proposed for harmonizing stability and competition in pair-based STDP. One solution is interpolating between hard and soft bounds to obtain a middle ground that can harbor both synaptic competition and stability, which is obtained over a limited parameter range [12]. Another solution, based on a small temporal shift in the STDP window, can stabilize the distribution of synaptic weights while maintaining competitiveness [13]. This shift has a similar effect in the triplet model [13]. To search the parameter space of the models for different stability/plasticity interplays, we systematically varied the balance between potentiation and depression parameters in each multi-spike STDP model. However, for each model, a fixed set of parameters was originally proposed to match experimental results. Our parameter changes may cause the response profile of the model to deviate from its originally fitted form. This can be justified because both the temporal spread and the magnitude of potentiation and depression vary considerably as a function of the location of a synapse [14,26,27]. Therefore, each parameter set in our analyses and numerical simulations could coincide with the characteristics of the STDP window at a particular location on the dendritic tree. Although all of the models we considered were proposed on the basis of experimental observations of synaptic modification, their effect on a population of synapses onto a postsynaptic neuron can be quite different. As mentioned above, one useful computational aspect of STDP is its ability to implement Hebbian learning and to functionally organize neural circuits. None of the three multi-spike models generated Hebbian competition when the original fitted parameters were used. Moreover, using these parameters, all three models produced an unstable distribution of weights tightly clustered near the upper bound of their allowed range. Given the observed broad distribution of synaptic weights in vitro [28,29,30] this is implausible. As it is possible to construct several phenomenological models that explain a given experimental data set, it seems reasonable to use the effects of plasticity at the population level (evaluated through simulations or analytical calculations) as a criterion for selecting a model. This criterion works particularly against model such as triplet STDP, because of its limited capacity for inducing competition among synapses at the population level even with altered parameter values, even though the model accounts for isolated experimental results satisfactorily. Finally a natural question is whether the STDP models we have considered are interrelated in any way, or whether it is possible to unite them in a single framework. The triplet and suppression models were motivated by different experimental data sets that showed opposite synaptic modification in response to triplets (ref. [6] vs. ref. [7]). However, the NMDAR-based model, which is phenomenologically closer to the molecular machinery involved in synaptic modification, can match the effects of either of these models, depending on the parameters used. Moreover, from the biophysical viewpoint, the other two models can be considered limiting versions of the NMDAR-based model through different simplifying assumptions about its components (Fig 10). If the second messengers activate instantaneously, the NMDAR-based model is qualitatively equivalent to the suppression model. The consumption of the limited pool of resting NMDARs by conversion into up or down states implements the suppressive effect of the preceding pre- or postsynaptic spike on the upcoming pre-post interaction. On the other hand, if there exists an infinite reservoir of resting NMDARs and inactive second messengers, the NMDAR-base model reduces to the triplet model. If both assumptions are fulfilled, the NMDAR-based model reduces to the simple pair-based model. This leads to the possibility that both the triplet and suppression models may arise from a single biophysical mechanism that involves NMDARs [31], but under different conditions for the speed of conformational changes and abundance of second messengers. We used a leaky integrate-and-fire (LIF) model neuron in our numerical simulations. The membrane potential of the LIF neuron obeys τmdVdt= (Vr−V) +Iex−Iin, (6) where τm is the membrane time constant, Vr is the resting potential, Iex is the excitatory input and Iin the inhibitory input. Although these inputs appear as currents, they are actually measured in units of the membrane potential (mV) because a factor of the membrane resistance has been absorbed into their definition. When the membrane potential V reaches the firing threshold Vth, the neuron fires an action potential and the membrane potential resets to the resting value Vr. The numerical values of all parameters are given Table 1. Each presynaptic action potential arriving at an excitatory or inhibitory synapse induces an instantaneous jump in the corresponding synaptic input (Iex or Iin), which decays exponentially between the input action potentials. The time course of the synaptic inputs can thus be expressed as Iex (t) =∑i=1Nexwi∑tik≤texp (tik−tτs), Iin (t) =win∑i=1Nin∑tik≤texp (tik−tτs), (7) where wi is the weight for excitatory synapse i, win is the common fixed weight for all Nin inhibitory synapses, and tik is the time of the k-th action potential at synapse i. The sums over presynaptic spike times are limited to spikes that arrive prior to the time t. The synaptic time constant τs = 5 ms is taken to be the same for excitatory and inhibitory synapses. The excitatory synaptic strengths, labeled collectively as w, are modified by STDP. If the rate of the excitatory and inhibitory inputs is rpre and rin respectively, the average firing rate of the LIF neuron can be approximated as [32] r¯post= (τmπ∫Vr−μσ+αVth−μσ+αdx exp (x2) (1+erf (x) ) ) −1, (8) where μ= (Nexrpre⟨w⟩−Ninrinwin) τsandσ2= (Nexrpre⟨w⟩2+Ninrinwin2) τs2τm, with ⟨w⟩ denoting the average value of the excitatory synaptic weights. The parameter α=|ζ (1/2) |τs/2τm, where ζ, is a correction to account for the nonzero synaptic decay constant. The arrival of a presynaptic spike increases the firing rate of the postsynaptic neuron transiently. For an LIF neuron in the case where the average excitatory input dominates over the inhibitory input, the firing rate after the arrival of a presynaptic spike at time t0 can be approximated as ([33]; see S1 Appendix) rpost (t) ≈r¯post+wexp (−t−t0τs) (Vth−Vr) τmΘ (t−t0), (9) where w is the strength of the synapse through which the presynaptic spike arrived, and Θ is the Heaviside step function. To study synaptic competition, we introduce correlations into half of the excitatory input spike trains. To generate Poisson spike trains with homogeneous pairwise (zero-lag) correlations, a “generating” spike train with rate r/c was first produced. The correlated spike trains were then obtained by trimming the generating spike train, that is, by randomly deleting spikes with probability 1 − c. The resulting spike trains all have rate r, and each pair is correlated with correlation coefficient c [34]. In pair-based STDP, a change of synaptic strength, Δw, is induced by a pair of pre- and postsynaptic action potentials with time difference (pairing interval) Δt = tpost − tpre. The functional relation between the synaptic modification and the pairing interval is Δw=F (Δt) ={A+exp (−Δt/τ+) ifΔt≥0−A−exp (Δt/τ−) ifΔt<0. (10) The positive parameters A+ and A− specify the maximum potentiation and depression, respectively. We express the synaptic strengths in units of the membrane potential (mV), so A+ and A− have mV units. The time constants τ+ and τ− determine the temporal spread of the STDP window for potentiation and depression (Fig 1A and 1E). In our analysis, we assume that the spike pairings are all-to-all, meaning that all possible pre-post pairs, not only the nearest neighbor pairs, contribute to plasticity. However, the results we derive apply qualitatively to a pair-based model with a nearest-neighbor restriction as well. In the triplet model, synapses are modified on the basis of pre-post pairing events in a manner similar to the pair-based model (Eq 10) but, in addition, when a synapse is potentiated by a pre-post pairing (Δt > 0), the postsynaptic potentiation variable Mpost is added to the amount of the pair-based potentiation A+. Similarly, when a synapse is depressed by a paring event (Δt < 0), the presynaptic depression variable Mpre is added to the pair-based depression A−. Thus, Δw=Ftrip (Δt) ={[A++Mpost (t−ϵ) ]exp (−Δt/τ+) ifΔt≥0−[A−+Mpre (t−ϵ) ]exp (Δt/τ−) ifΔt<0. (11) The small parameter ϵ ensures that the values of Mpre and Mpost just before their update by the pre- or postsynaptic spikes are used. The postsynaptic potentiation and presynaptic depression variables are governed by the equations dMpredt=−Mpreτpre+Apre∑iδ (t−tpre (i) ) dMpostdt=−Mpostτpost+Apost∑iδ (t−tpost (i) ), (12) where δ (t) is the Dirac delta function, and tpre (i) and tpost (i) are the times of arrival of pre- and postsynaptic spikes respectively. This introduces four parameters into the model beyond those of the pair-based model: the time constants τpre and τpost and the increments Apre and Apost. In the suppression model with time constants τpre and τpost, the change in a synaptic weight is determined by Δw=Fsupp (Δt) =[1−exp (−Δtpre/τpre) ][1−exp (−Δtpost/τpost) ]×{A+exp (−Δt/τ+) ifΔt≥0−A−exp (Δt/τ−) ifΔt<0, (13) where Δtpre is the interval between the presynaptic spike in the pair and its preceding presynaptic spike, and Δtpost is the interval between the postsynaptic spike and its preceding spike. The suppression model introduces two new parameters beyond those of the pair-based model: the time constants τpre and τpost. The NMDAR-based model [16,18] is based on the assumption that NMDARs can be in one of three different states: “rest”, “up” and “down”. The variables frest, fup and fdn denote the fraction of NMDARs in each state respectively (frest + fup + fdn = 1). In the absence of pre- and postsynaptic spikes, the receptors in up and down states return to the rest state with time constants τfup and τfdn respectively. Each presynaptic spike up-regulates the receptors immediately after its arrival by an amount proportional to a parameter Afup, and each postsynaptic spike down-regulates the receptors proportional to a parameter Afdn. The dynamics of the NMDARs in the “up” and “down” states can be expressed as: dfupdt=−fupτfup+Afupfrest∑iδ (t−tpre (i) ) dfdndt=−fdnτfdn+Afdnfrest∑iδ (t−tpost (i) ), (14) where the sums run over all pre- (tpre (i) ) or postsynaptic (tpost (i) ) spike times, indexed by i. In this and subsequent equations, we assume the convention that a quantity multiplying a δ function is evaluated immediately before the time when the argument of the δ function is zero. The fraction of active second messenger Mup is increased by postsynaptic spikes proportional to the amount of up-regulated NMDARs fup and the available inactive messengers 1 − Mup. Likewise, the fraction of active second messenger Mdn is increased by presynaptic spikes proportional to the amount of down-regulated NMDARs fdn and available inactive messenger 1 − Mdn. In the absence of spikes, these second messenger fractions decay with time constants τMup and τMdn, respectively. Thus, dMupdt=−MupτMup+AMupfup (1−Mup) ∑iδ (t−tpost (i) ) dMdndt=−MdnτMdn+AMdnfdn (1−Mdn) ∑iδ (t−tpre (i) ), (15) where the sums run over all pre- (tpre (i) ) or postsynaptic (tpost (i) ) spike times. The parameters AMup and AMdn governing the magnitude of the changes in the messengers on spiking events. Finally, synaptic potentiation occurs in response to postsynaptic spikes and depends on the amount of Mup, and synaptic depression occurs in response to presynaptic spikes depending on the amount of Mdn, so that dwdt=A+[Mup−θup]+∑iδ (t−tpost (i) −ϵ) −A−[Mdn−θdn]+∑iδ (t−tpre (i) −ϵ), (16) where θup and θdn are thresholds above which the corresponding messengers take part in plasticity, and [x]+ denotes the piece-wise linear threshold function [x]+ = x for x > 0 and zero otherwise. The small parameter ϵ is included because, in this case, we evaluate the factors multiplying the δ functions after the time of a spike, as required by the model. | Title: Stability and Competition in Multi-spike Models of Spike-Timing Dependent Plasticity Summary: Synaptic plasticity is believed to underlie learning and memory by competitive strengthening and weakening of synapses in neural networks. However, the ability to form new memories while maintaining the old ones involves an intricate balance between synaptic stability and competition. In one of the most widespread such mechanisms, spike-timing dependent plasticity (STDP), the temporal order of pre- and postsynaptic spiking across a synapse determines whether it is strengthened or weakened. Early description of STDP only took into account pairs of pre- and postsynaptic spikes. However, more recent experimental results showed that the "pair-based" description is not sufficient to fully account for synaptic modifications under STDP, and motivated more complex "multi-spike" STDP models. While the conditions under which the pair-based STDP leads to synaptic stability and/or competition are well studied, it is not clear when and how multi-spike STDP models lead to synaptic stability and competition. Here, we address these questions through numerical simulation and analysis of a population of plastic excitatory synapses that converge to a neuron. We show that different multi-spike STDP models can induce synaptic stability and competition under radically different conditions, which have important implications in relating learning and memory to biophysical properties of synapses. | 12,988 | 307 | lay_plos | en |
Summarize: This invention relates to urinary catheters, and in particular to a method and apparatus for a pressure-activated, magnetic-force-controlled human (or animal) bladder drainage cycling valve system(Uro Cycler), for restoring normal body functions of bladder filling and emptying in cyclic manners of catheterized patients, and this invention claims the benefit of priority to U.S. Provisional Application No. 60/280,767, filed Apr. 2, 2001, U.S. Provisional Application No. 60/280,768 filed Apr. 2, 2001, and U.S. Provisional Application No. 60/324,601 filed Sep. 25, 2001. BACKGROUND AND PRIOR ART Urinary catheters bypass the normal bladder process of storing urine, and only releasing the urine by using the bladder detrusor muscle. Catheters can be a necessary tool to open the bladder to allow urination when patients have trouble urinating. A catheter can be a lifesaving tool since an uncontrolled buildup of urine can cause serious medical problems including death. However, there are known problems with catheters. Struvite crystal encrustation is the effect of stagnated urine in the neck of the bladder when using a catheter. In the face of an indwelling catheter, urine can pool at the neck of the bladder, and the pooled urine can shift from a normal pH factor to an abnormal pH level of 10 or more while it stagnates. Urine shifts to an ammonia state where struvite crystals can precipitate and enlarge on the indwelling catheter. This situation can occur as the bladder loses its natural ability to cyclically flush itself in the face of an indwelling catheter. Bladder wall thickening has also be observed in long-term catheterizations and may be a result of the increasing pH levels. Urinary tract infections can occur as the urine stagnates and shifts from its normal, acidic antibiotic property through the pH spectrum. Pooled urine that can occur in the neck of the bladder beneath the indwelling catheter can be a natural breeding ground for microbes which can migrate in the body. Bladder spasms can also occur with an indwelling catheter which causes the bladder to cease its normal cycle of filling and flushing. A dynamic functioning system is converted to a static state with a catheter, and painful bladder spasms can occur. Bladder atone can also occur where short term or more permanent loss of natural bladder functions occurs by using a catheter. It is also generally well known in medical circles that a human bodies primary defense mechanism against urinary track infections and the other problems listed above is the process known as “wash-out”, where it is advantageous to allow a bladder to normally fill up and be released at one time rather than in an uncontrolled drip fashion that would occur with using a catheter. Various catheter type instruments and procedures have been used for draining bladders of patients in hospitals. These instruments and procedures have evolved from constant (non-cycling) drip drainage through painfully inserted catheters by siphoning, suction and various types of awkward manually externally controlled cycling apparatus and procedures. Fundamental to an effective, safe, and appropriate device and method is allowing the bladder to fill reasonably and then draining it without a suction pump and without allowing build-up or entry of infectious contaminants in the drainage system. Included in previous methods and devices have been U.S Pat. Nos. 2,602,448 and 2,860,636 which utilized a siphon in combination with a reservoir to provide cyclic draining of the bladder. Pressure release in these devices is controlled by raising the height of the device on a bedside tree. It is subject to distortion by shifting and turning of the patient and is unreliable (and can compromise safety) besides restricting the patient. U.S. Pat. No. 3,598,124, describes a siphon leg controlled by merely attaching a catheter to a bedside tree at predetermined adjusted height, which varies the pressure at which the bladder will drain and provides a flutter valve near the patient to break the siphon action of the system once the bladder has drained. U.S. Pat. No. 4,230,102, describes a device for the draining of a bladder in which a T-joint has been placed on a catheter and has a pressure membrane attached thereto in a large casing for actuating a pressure switch which in turn actuates an electric motor driving a gear train and cam. A cam follower is spring loaded to clamp the catheter for two minute cycles upon actuation by the pressure switch to drain the bladder. However, this type of device, can be expensive and bulky and positions an electrical apparatus adjacent to the catheter. U.S. Pat. No. 4,424,058, describes a spring-return valve in conjunction with a siphon-release orifice to prevent excessive suction and to prevent urine from remaining in the system after drainage. A problem with this system was that the restoring force of the spring increased with distance of travel from a closed position. This valve was very unsatisfactory because it closed again as soon as the urine fluid pressure dropped off, thus causing fluid to remain trapped in the bladder to stagnate with further elapsed time. Only a full bladder would open it, sometimes at an uncomfortably high (and potentially unsafe) pressure, and only a relatively full bladder would keep it open to allow complete drainage unless overridden by the patient bearing down heavily on the lower abdomen. Also, positioning of tubes leading from it was parallel to the leg on which it was attached and provided a situation for retention of fluid in the system. None of the proposed patented devices and techniques described above solves all the problems with catheters that are listed above. Unlike the problem methods and devices of the prior art, the subject invention provides both consistent magnetic opening and closing of a valve seal with decreased rather than increased closing pressure when opened. As the bladder is emptied, decreasing head pressure against the valve can keep the valve open to establish complete and sterile drainage. In addition, the successfully-tested clinical embodiment of this invention provides simple and convenient manual override, when desired, to decrease or eliminate totally the magnetic closing pressure of the valve. SUMMARY OF THE INVENTION A primary objective of the invention is to provide a low pressure magnetic valve for bladder management cyclic flow control having consistent opening and closing of the valve seal with decreasing head pressure against the valve as opposed to increasing pressure. As long as any fluid is coming through the line, the valve will remain open until a complete emptying of the bladder is achieved. A secondary objective of the invention is to provide a low pressure magnetic valve for bladder management cyclic flow control that establishes complete and sterile drainage as the bladder is being emptied. A third objective of the invention is to provide a low pressure magnetic valve for bladder management cyclic flow control that can be automatically run with a simple and convenient manual override that can be selectively engaged. A fourth objective of the invention is to provide a low pressure magnetic valve for bladder management cyclic flow control that helps restore normal body functions of bladder filling and emptying in a cyclic manner, with normal, healthy pressure sensations in spite of the presence of the catheter which typically inhibits “natural” bladder drainage. The fifth objective of the invention is to provide a low pressure magnetic valve for bladder management cyclic flow control for use with a catheter which can reduce and eliminate known problems that occur with using a catheter such as urinary tract infections, struvite crystal encrustation, bladder spasms and bladder atone. The sixth objective of the invention is to allow a user wearing a catheter to use their bladder detrusor muscle to selectively turn on a valve in the catheter and complete an entire urination emptying cycle of their bladder. The invention provides for both consistent magnetic opening and closing of a valve seal with decreased rather than increased closing pressure when being opened. As the bladder is being emptied, the decreasing of head pressure against the valve can keep the valve open to establish a complete and sterile drainage. In the invention, valve-closing pressure can decrease as a result of three important factors: (1) magnetic pull of a valve decreases as its open distance from magnetic attraction in the direction of the valve seat increases, (2) the gravity-enhanced fluid flow column in the drain down tube provides a slight negative pressure on the back side of the movable magnet (thus tending to hold the valve open until the drain tube empties completely), and (3) fluid passing through the system provides a partial mass flow insulation which tends to hold the moving magnet away from the fixed magnet, also decreasing the net magnetic attraction between the magnetic members. A small amount of air is allowed to leak through a micro-pore filter (which keeps out harmful micro-organisms from the closed system) in order to vent the down line for clean, dry emptying. The very low-pressure valve system of the invention for use in controlling the flow of most fluids utilizes magnetic forces to hold a smooth surface against an ultra low-durometer (soft) composite seal or valve seat material until such time as the fluid head pressure causes the magnets to separate and the valve to open, at a preset value, to allow maximum fluid flow rate and complete drainage of the system The use method described here is medical in nature, applying to bladder drainage of catheterized patients into a urine collection bag, as needed, in a normal, cyclic fashion. In other words, head pressure of urine building up in volume against the detrusor muscle of a bladder and in a catheter running from the bladder to the valve where it is positioned on a patient's leg or rests on the bed sheet, causes the valve to open away from the valve-port seat. When the valve is opened, distance increases between the valve magnetic member and a member to which it is magnetically attracted in the direction of the valve-port wall, thereby allowing the valve to remain open with less pressure than that initially required to open it. Fluid passing between the open valve and the member to which it is attracted magnetically decreases further still the closing pressure to offset the head-pressure opening of the valve. Downstream from the valve, there is a siphon-release air-inlet orifice that relieves siphon (negative pressure) to avoid siphon suction that would either cause collapse of the bladder walls or cause the valve to remain open after the bladder is emptied. An air inlet, allowing only air flow through a micro-pore filter material to the siphon-release orifice, is positioned upstream and radially outward from an outlet to the valve in order to prevent passage of fluid from the valve where siphon pressure does not provide sufficient inward suction of air. The siphon-release orifice is provided with an antiseptic strainer (anti-bacterial/anti-viral filter) and can serve as a low-pressure one-way inlet valve. The entire valve system (in the embodiment of a small, streamlined, compact, integrated and durable device) also serves as an anti-reflux valve between the patient and the urine collection bag, thus preventing drained (and possibly old and unsterile, septic, contaminated) urine from ever re-entering the catheter, urethra, and bladder of the patient, and potentially causing infection or other problems. Additional embodiments of this invention provide for a manual override of the valve by selective distancing an externally positioned magnetic member from the valve member that is attracted to it. The override gives flexibility of pressure adjustment and provides the opportunity of assuring full drainage when desired by either physician or the patient. This could manifest itself, in the event of excessive discharge of viscous matter or other mode of lumen blockage, as a “safety” valve to relieve fluid pressure buildup. An additional swivelable attachment of the bladder cycler to a strap on a patient's leg can allow the cycler to be positioned comfortably at a slant with the outlet and tubes leading from it downward from the valve to further assure that fluid will not remain in the system between drainage cycles whether used in either a prone or vertical position of the leg. The patient also can more readily move about and not be confined or attached to the bed as long as the collection bag is kept attached for use as needed. The invention can be used as a hospital instrument whenever an indwelling catheter is required, or in clinics, or in physician's offices, or in homes for draining urine from bladders of patients automatically and safely after normal filling, thoroughly and antiseptically. This use is in strong contrast to the typical, non-cyclical, continuous drip associated with urethral catheter drainage into a collection bag. The use of the UroCycler with catheterized patients helps restore the more normal body function of bladder filling and emptying in a cyclic manner, with normal, healthy pressure sensations in spite of the presence of the catheter which here-to-fore prevented “natural” bladder drainage. Further objects and advantages of this invention will be apparent from the following detailed description of the presently preferred embodiments which are illustrated schematically in the accompanying drawings. BRIEF DESCRIPTION OF THE FIGURES FIG. 1 is a perspective view of a fully assembled, automatic drainage embodiment. FIG. 2 is an exploded perspective view showing the components comprising this passive magnetic bladder-cycling valve. FIG. 3 is a cutaway of the assembly showing the valve in the closed configuration FIG. 4 is a cutaway of the assembly with the valve open. FIG. 5 is a section view of the assembly showing the valve closed. FIGS. 6A and 6B shows an added over-pressure safety release plug embodiment. FIG. 7A is a section view of assembly with the valve open. FIG. 7B is an enlarged view of an optional screw with magnet that can alter valve opening pressure. FIGS. 8A, 8 B, 8 C, and 8 D show embodiments of valve seal and valve seat combinations and useful configurations. FIGS. 9A, 9 B, 9 C and 9 D shows a fluid pressure and flow time chart showing the invention system operation. FIG. 10 illustrates the bladder cycler being used with a connected clinician's (or physician's) self-sealing sampling port on the inlet end and a hydrodynamically-balanced outlet downline to a fluid collection bag on the outlet end. FIG. 11 is a side view of a patient's leg with the invention strapped to it. DESCRIPTION OF THE PREFERRED EMBODIMENTS Before explaining the disclosed embodiments of the present invention in detail it is to be understood that the invention is not limited in its application to the details of the particular arrangement shown since the invention is capable of other embodiments. Also, the terminology used herein is for the purpose of description and not of limitation. FIG. 1 is a perspective view of the fully assembled bladder cycling passive magnetic valve invention 1. FIG. 2 is an exploded view of the internal components of the invention 1 of FIG. 1. Referring to FIGS. 1 and 2, this exploded view shows the key components comprising the assembly. The inlet end, or upper non-magnetic housing 2 has female socket end 2. 2 which mates with the male prong end 3. 2 of outlet end, or lower housing non-magnetic 3, with a water-tight and air-tight bond. A moving magnetic valve member 8 is magnetically attracted in the direction of valve-port wall 4 where the resilient valve seal 9 contacts the valve seat at the outlet end of valve insert orifice 4. The moving magnetic valve member 8 is attracted magnetically to the valve-port wall 4 due to the fixed upstream magnetic member 7. Inwardly protruding rails 3. 6 in lower housing 3 form an internal chamber for allowing the moving magnet valve 8 to slide back and forth. Fixed magnetic member 7 is held in place by prongs 4. 6 extending from wall 4 having openings/slots 4. 3 therebetween. The openings/slots 4. 3 are large enough to allow for fluid flow around the fixed magnetic member 7. Magnetic attraction can be provided by composition of either or both the moving valve member and the upstream magnetic member. Optionally and preferably, the magnetic valve member 8 and the upstream magnetic member 7 both are inert, ceramic permanent magnets having high magnetic field saturation and high coercive magnetic force, and the valve-port wall 4 is non-magnetic. Opposite, attracting poles of each magnetic member 7, 8 are facing each other in the fully operational valve. However, when double-blind studies are conducted, a fully assembled “dummy” valve can be substituted with like magnetic poles facing each other, thus making a non-closing, constantly open, “placebo” unit. For testing purposes, a preferred embodiment of the moveable magnet member 8 was measured at approximately 130 milligauss(mG) at a distance of approximately 7 millimeters(mm) from the sensing coil of a DC Magnetometer. The fixed magnet member 7 had a measurement of approximately 40 mG. Both magnet members 7, 8 were approximately 0.375 inches in diameter. For the preferred embodiment, the pressure needed to open the valve member 8 away from its seat was adjusted to be approximately 0.1 ounces per square feet(ounces/sq.ft), which correlated to approximately 15 cm height of H2O, in the catheter line. Referring to FIGS. 2, and 5, channel ridges 17 at the inside periphery of non-magnetic housing 3 provide fluid passage linearly between them from side-to-side of first the magnetic base member 7 and then the magnetic valve member 8. Referring to FIGS. 5, and 7 A, stopper shoulders 18 are provided to arrest travel of the magnetic valve member 8 at a select distance of travel away from valve-port wall 3. 4. Referring to FIGS. 5, 7 A and 8 A- 8 D, the valve-port wall is provided with a valve-seat ridge 9 for reduction of valve-seat area to reduce area for accumulation of particulates in fluid passing through the system and for providing a relatively smaller surface for tightly seating into the moving valve member 8. Moving valve member 8 can move in an interior chamber within housing section 3 in both directions as shown by arrows M 1 and M 2. A resilient non-magnetic valve surface 9 can be provided for increased seating pressure and for selectively decreased magnetic attraction in the direction of the valve-port wall 19. 2. Alternative valve seat/seal configurations are illustrated in FIGS. 8B, 8 C, and 8 D. In FIG. 8C, the low-durometer, soft, resilient seal 4. 2 can be built into the insert 4 member and its sealing ridge 19, and a smooth surface of magnet 8 can press firmly enough to make a suitable urine flow seal. In FIGS. 8B and 8D, an FDA-approved material (eg: silicone) resilient O-ring 15 can be either bonded or inserted in the wall member of valve seat insert 4 to provide a low-pressure fluid seal against the face of the moving magnetic material 8. Referring to FIGS. 1 through 5, and FIGS. 7 A and 8 A- 8 D, inside corners of magnetic valve member 8, inside corner edges of housing inlets 2, inside corner edges of housing outlets 3 and all other corners possible can be rounded to facilitate flow through the system and to prevent accumulation of particulates in fluid passing through the system. Outside surfaces of inlet housing 2 and outside surface of outlet housing 3 also can be rounded to prevent scraping action that would tend to accumulate particles at the outside and decrease cleanliness. In addition to being rounded, the inside corners of the housing outlets can be angled from the basically symmetrical barbed inlet and outlet connectors 11 which can be selectively tapered, ribbed or otherwise designed to receive and to hold medical tubing. Referring to FIGS. 1 through 7B, an optional soft elastic plug 12 may be inserted into a vent line 13. 2 / 13. 4 to serve as a safety pressure release valve (perhaps if blockage in the system caused urine pressure in the bladder to build up past some potentially hazardous number like 80-120 cm/H2O column) which would pop out to avoid renal or other physiological damage. Details are shown in FIGS. 6A and 6B. In addition, this feature could allow medicine to be injected into the urinary tract on the bladder side, if desired, to control or prevent infection. FIG. 6A has a blunt edge plug 12. 2, which fits within a uniform diameter vent line 13. 2, and an alternative version FIG. 6B has an expanded tip 13. 4 which fits within and catches against an interior surface about narrowing vent line 13. 4 Referring to FIG. 7B, another (threaded) hole 2. 5 could be used to position a screw 29 /magnet 30 mechanism so as to position a third permanent magnet 30 in close proximity to the back of the “fixed” magnet member 7, in order to alter the net magnetic field strength and thus control the valve opening pressure. The present magnet-holding insert 4 can be molded in a soft plastic material in order to make a good seal against the moving magnet face on component 8. As shown in FIG. 7A, the South Pole of the moving magnet 8 faces the valve seat 19 at its left. This South Pole is attracted to the North Pole of the fixed magnet 7 behind the seat 19 to its left. The manual external rotational adjustment of externally adjustable screw 30 controls the magnet 30 South pole separation from the magnet 7 South pole which would allow a significant degree of valve pressure opening adjustment, or variable pressure setting, which can be desirable in certain situations. The closer magnet 30 is to magnet 7, the less net field strength there is to attract magnet 8. Conversely, when the screw 29 is backed off so that magnet 30 is farther apart from magnet 7, then magnet 30 has less effect in canceling some of the strength of magnet 7, and the opening pressure is higher. When bladder detrusor muscle atony has occurred, or when therapeutic bladder retraining is called for, lowered position of magnet 30 toward magnet 7 (screwing the magnet 30 in closer), or graduated pressure settings can provide substantial additional benefits. Currently, the opening pressure setting can be determined by the insert dimensions (assuming a consistent magnet gauss reading) and each unit can acquire a fixed pressure value during the manufacturing process. While the normal opening range is approximately 15 to approximately 30 cm/H2O, individual units can be made to operate at higher or lower pressures, within practical limits. Head pressure to open the valve can be decreased by pressing the button inwardly and sliding the magnetic base member in the direction of the housing inlet. The valve can be totally released without any magnetic pressure to hold the valve shut when the magnetic base is slid to the extreme housing-inlet end of travel of the button stem in the stem channels. Closing pressure of the valve is increased by sliding the magnetic base member in a downstream direction toward the housing outlet. Referring to FIGS. 2, 3, 4, 5, 7 A, 7 B and 10 a siphon air vent micro-port filter 6 and cover 5 can be provided to effect a very low-pressure siphon, suction, or negative pressure from fluid passing through the housing outlets 11. 2 and down the drain line 22 into the collection bag 27. Typically the filter material within filter 6, either woven or non-woven attaches by an adhesive and is packed around the housing vent hole 10. This feature of the invention assists in holding the valve 8 open until all the fluid is emptied out of the system as well as allowing the system tubing to drain clean and dry, thus preventing moist surfaces within the system for potential bacterial growth. The vent filter 6 allows for a stream of air bubbles to enter line 22 to aid in allowing complete drainage of all fluid through line 22, since in a gravity directed flow system an upper located vent enhances fluid flow therethrough. Typically for construction purposes, the vent valve 10 can be positioned at the outside periphery of a valve-port wall 3. 6 and the vent aperture 10 can be positioned in the outside periphery of an outlet housing member 3 that is created during plastic injection molding process or in the construction assembly process. The assembly can be either glued, chemically welded, ultrasonically welded, or press-fit snugly enough to remain assembled without glue. Referring to FIG. 2, the magnetic base member 7 can be either glued or otherwise fixed in seats 4. 6 in a position at a select distance from the valve-port wall (seat) to achieve a pre-determined pressure requirement for opening of the valve 8 in opposition to magnetic attraction of the base member 7 and the valve 8. Alternatively, however, the magnetic base member 7 in FIG. 2 can be moved by an automatic, but very weak, drainage spring or other weakly resilient member. When pressure from the weight of fluid in the bladder 23 (shown in FIG. 10) and in the column from the bladder 23 to the bladder cycler invention 1 cause the valve 8 to open in opposition to the magnetic attraction, the spring action will cause the base member 7 to move upstream away from the moving valve member 8 and thereby decrease further the attraction between the two magnets 7, 8. This allows more complete emptying of the bladder contents 23. 5. Although not manually-controllable, this invention provides some features of the controllable embodiment at a lower cost of construction. A springy material in this working relationship functions in the opposite direction as springs used to close valves in prior-art practices. It decreases rather than increases opening pressure of the valve when pressure in the bladder is low from being partially emptied. This configuration would require a very careful design and implementation in order to balance the static and dynamic forces precisely for operation, both in the opening pressure and to assure valve closing, sealing without leaking during urine pressure buildup to the opening threshold. Referring to FIG. 10, this diagram traces out the cycler invention use method as the key component of a hydrodynamically-balanced cyclic urinary drainage system. The human (or animal) bladder 23 and bladder contents 23. 5 with its two ureter inputs has its attached urethra 24 invaded by an indwelling catheter (such as a balloon-anchored Foley type) 25. On the output end, this catheter can be connected to a clinician's (physician's) sampling port 26, from which a urine sample can be drawn by either a conventional syringe needle or by a safety plastic canula probe. Operation of using the cycler will now be described in reference to FIGS. 2, 3, 4, 5, 7 A and 10. Initially, the valve 8 is in a closed position to inhibit fluid flow therethrough. Arrow 14 A of FIG. 3 shows the direction of fluid flow which stops by closed seated valve 8. A patient can use their bladder detrusor muscle 23. 7 about the bladder 23 to cause a small amount of pressure in the catheter line 25, 22 to cause the valve 8 in cycler 1 to pass to an open position(in the direction of arrow M 1 ) to allow fluid flow therethrough. Arrows 14 B and 14 C of FIG. 4 show fluid passing through cycler 1. Fluid running down line 22 assists in maintaining the valve 8 in an open position by causing a hydrodynamic pulling on the valve 8 so that all fluid flow passes therethrough. The hydrodynamic pulling on the valve 8 causes by the fluid flowing through line 22 is stronger than the attractive forces between magnet valve 8 and member 7. After all fluid passes through line 22, no further hydrodynamic forces exist to keep the valve 8 in the open position so now valve 8 is free to move in the direction of arrow M 2 to a closed position since the attractive power of the magnets 7 and 8 causes valve 8 to move in the direction of arrow M 2. Unlike using actual spring biased-backed valves used in some prior art devices, the subject invention valve 8 does not function in an equivalent manner. For example, the larger the opening of the magnetic valve 8, the less the pull(magnetic attraction with member 8 ) exists to close the valve 8. With a spring backed valve, the greater the opening in a valve, the greater the resistance is from the compressed spring to cause a closure of the valve. For example, a patient exerting fluid pressure to open a spring biased-backed valve has greater resistance that occurs as they try to increase urination pressure. With the subject invention magnetic valve 8, more pressure from the bladder causes less resistance against the valve 8, and helps fluid flow therethrough. Additionally, spring biased-backed valves have been known to cause a premature closing in a catheter line which can cause any of the medical problems referred to in the background section of the invention. The subject invention magnetic valve 8 does not prematurely close since the hydrodynamic gravity enhanced pulling of fluid downline is enough to overcome the magnetic attraction to keep the valve open. When downline fluid flow ceases, the hydrodynamic gravity enhanced pulling of fluid ceases and the magnetic attraction is enough to close off valve 8. The subject invention allows a person to use their bladder detrusor muscle to cause pressure selectively turn on and complete urination emptying cycle of the contents of the bladder while wearing a catheter. The cycler 1 can be an alternative to using dangerous clamps on a catheter line, since clamps left on a catheter line for extended periods of time can be hazardous to a patient's life. The cycler 1 allows for a fluid samples to accumulate about port 26 so that an adequate sample can be retrieved when needed. Referring to FIG. 10, the cycler invention 1 with magnetic moving valve 8 can be connected between the sampling port 26 and the specially-sized downline tubing 22, thus forming a hydrodynamically-balanced drainage system, terminated by the collection bag 27 and its clamped-off emptying tube 28. A preferred embodiment incorporates the connections from the catheter be made as shown, and that the downline from the outlet end of the bladder cycler valve consist of approximately 0.1875 inch inside diameter tubing, preferably of polyethylene construction. The surface tension of the watery urine fluid 23. 5, combined with the lumen of the catheter and the fluid pressure, fluid flow rate, viscosity, and gravitational forces have shown that these dimensions, including the length of the drainage line, can be critical parameters for proper and safe, reliable operation as a listed Class II, U.S.F.D.A.(United States Food and Drug Administration) 510(k) medical devices. As previously described, for the preferred embodiment, the pressure needed to open the valve member 8 away from its seat was adjusted to be approximately 0.1 ounces per square feet(ounces/sq.ft), which correlated to approximately 15 cm height of H2O, in the catheter line. Referring to FIG. 11, a leg strap 20 can be provided about a human leg 20. 5 with a swivel connection 21 that allows the bladder cycler invention 1 to be positioned when desired at a downward angle with respect to a leg 20. 5 to which it attached. This allows a catheter or outlet drainage tubing 22 to be positioned at a slant that provides downward flow of fluid that otherwise could remain in the system between drainage cycles. Referring to FIGS. 9A, 9 B, 9 C and 9 D, these graphs show the time relationship between pressure buildup on a test stand, such as in the configuration diagrammed in FIGS. 10-11, the valve opening pressure drop, the initiation of fluid flow, the constant flow rate until the point of emptying, the valve closing again, and the cycling period to the next pressure fill and open sequence. This period is normally approximately two to approximately four hours for the average adult, depending on amount of fluid beverages consumed, physical activity, and physiological factors such as bladder size, general health, pressure sensitivity, and the like. In FIG. 9A, the commencement of urine flow is indicated at point 60. The flow is quite consistent throughout the time period ending at point 61, about 60 seconds. The flow charted from point 62 to 63 represents another bladder emptying cycle. FIG. 9B illustrates the bladder pressure buildup cycle from 17 cm of water column pressure at point 70 to the valve opening threshold pressure of 21 cm at point 71, dropping to zero at point 72. At point 73, the chart stops and a new partial cycle from point 74 at 16 cm of H2O to the opening point 75 at 22 cm of H2O. FIGS. 9C and 9D shows charts that indicate that the flow rates and urine volumes are in the “normal” range(e.g. approximately 250 cc over 60 minutes. Referring to FIG. 7A a manual override embodiment can allow for selectively keeping valve 8 in an open position. For example, an extra outside magnet 50 can be positioned adjacent filter 6 to have South pole S, that attracts North pole N, of moving valve 8 in an open position. The manual override of the valve can occur by selective distancing of an externally positioned magnetic 50 from the valve 8 that is attracted to it. The override gives flexibility of pressure adjustment and provides the opportunity of assuring full drainage when desired by either physician or the patient. This could manifest itself, in the event of excessive discharge of viscous matter or other mode of lumen blockage, as a welcome “safety” valve to relieve fluid pressure buildup in the line and system upstream from the cycler 1. While the invention has been described, disclosed, illustrated and shown in various terms of certain embodiments or modifications which it has presumed in practice, the scope of the invention is not intended to be, nor should it be deemed to be, limited thereby and such other modifications or embodiments as may be suggested by the teachings herein are particularly reserved especially as they fall within the breadth and scope of the claims here appended. | Summary: A low-pressure fluid flow control magnetic valve (cycler) device, system and method for use emptying the contents of bladder by a catheter. The device can be connected with a specified drainage tubing to function as a hydrodynamically balanced system (to empty into a typical two-liter collection bag). The cycler can be connected externally to a urinary catheter for hospital, clinical and home-care use for the emptying of the bladder of a patient through a catheter in a biologically more natural, filling and draining, cyclic manner. Fully automatic, modes of operation are provided for opening and closing this valve to empty the bladder of urine when appropriate or necessary. The modes of operation respond to normal human (or animal) body pressures, or are automatic with a manual override. This device is U.S.F.D.A. approved for human use and responds to normal human (or animal) body pressures to assist the detrusor muscle to function normally in spite of an indwelling catheter challenge. The cycler device avoids problems with bladder atone, bladder spasms, harmful struvite crystal formation, bladder retraining after surgery, and allowing urine tract washout to occur as the body's primary defense mechanism against urinary tract infections. | 8,116 | 283 | big_patent | en |
Summarize: “The images tell us all that MeerKAT is the best telescope of its kind in the southern hemisphere, with only 16 dishes,” he told ministers, deputy ministers and visitors gathered at the site of the Square Kilometre Array (SKA) in the Northern Cape on Saturrday. Durban - The first image from MeerKAT shows that the telescope “will be a remarkable discovery machine”, the project’s chief scientist, Dr Fernando Camilo, says. Early results show the MeerKAT radio telescope in the Northern Cape is destined to be a powerful research tool, scientists say. Early results show the MeerKAT radio telescope in the Northern Cape is destined to be a powerful research tool, scientists say. Credit: SUPPLIED Science and Technology Minister Naledi Pandor inaugurated the first 16 dishes of the MeerKAT telescope - and unveiled its first image. By the end of next year, the South African-designed and built telescope will comprise 64 dishes. These giant two-storey dishes rise up out of the centre of the MeerKAT core area, standing incongruously against a backdrop of ocre sand and scrubland. Others wait to be assembled, their white metal bowls ready to be hoisted on to pedestals. “When the full 64-dish MeerKAT is available, it will be the best telescope of its kind in the world,” said Camilo, who left Columbia University, New York, to take up the post in April. It was difficult to get to this stage as there was usually a need for trouble-shooting before a telescope could produce a high-quality image. “(The image) tells us and the world that we have a working telescope in the Karoo,” Camilo said. Although the R3 billion MeerKAT is owned and funded by South Africa, it will be incorporated into the SKA, which is to be the largest telescope in the world. The SKA is an international project and will be hosted by Australia and South Africa, with satellite sites in eight African partner countries. It will seek to answer some of humanity’s most enigmatic questions: Are we alone in the universe, what is dark matter, how do galaxies evolve, and what happened after the Big Bang? From 2018, another 133 dishes will be added to MeerKAT to form part of the phase one of the SKA, the cost of which has been capped at R10bn. In Australia, about 130 dipole antennas, which look like 2m Christmas trees made out of thick wire, will be constructed as part of this phase. South Africa decided to build MeerKAT even before it was announced in 2012 that the SKA would be split between the two countries. This was in part to showcase South Africa’s scientific and engineering capabilities and prove the country could build and host a radio telescope. The intention was also to ensure it would have a legacy project in case the country lost the hosting bid. “This is not just an unveiling,” Pandor said yesterday. “We want to show the world the kind of research that the MeerKAT-16 makes possible... We were meant to reach this (quality of image) at 32 (dishes), not 16.” It is difficult and expensive to engineer a radio telescope with a single big dish. This is why the MeerKAT - and ultimately the SKA - is an interferometer, which uses many smaller dishes acting as one giant telescope. This means an interferometer can be brought into use in phases and the radio telescope can be used for scientific work before all phases have been completed. Professor Justin Jonas, SKA South Africa’s chief technologist, said he was “amazed” at the quality of the image. He added, however: “There are no accidents here. It’s been a coherent effort from the whole team and the National Research Foundation and the Department of Science and Technology... We hired the right people, had the right processes in place. “Personally, I’m very, very excited. I’ve been wanting to build a radio telescope since I was a kid, and now we have. How many people get to do that? And it’s working!” The released image was a picture of about 1 300 radio galaxies, of which only 70 had been imaged before, said Jonas, who attended the first international SKA meeting as South Africa’s ambassador. Celestial objects, like stars and galaxies, emit radio waves, which cannot be seen with the naked eye. Radio telescopes receive these fairly weak signals from the universe and turn them into maps and images of the universe. The radio spectrum is substantially broader than that of visible light, which means scientists can “see” more with radio telescopes. The “first light” image was taken in the L-band, which is a portion of the radio spectrum. This band is of interest globally because it contains information about how galaxies and the universe evolve, among other things. MeerKAT is expected to be twice as sensitive in this band as was initially anticipated. This means an experiment in this band could take a quarter of the time that has been allocated. Pandor said there was more to South Africa’s astronomy investment than pure science: “Big science brings opportunity to South Africa and the African continent. “The SKA brings opportunities to this area, opportunities they hadn’t hoped for.” It was “not always easy to convince governments to support long-term projects and initiatives, especially in science… (where) there are often nuanced impacts that are not immediately visible”. Pandor encouraged Economic Development Minister Ebrahim Patel, 11 deputy ministers and the other dignitaries present to be “ambassadors for science and the SKA”. “Science and astronomy science can change lives, change communities, build human capital. “Through science, in South Africa and Africa, we are able to advance development.” Wild was a guest of the Department of Science and Technology and SKA South Africa. Sunday Tribune (CNN) A South African radio telescope has revealed hundreds of galaxies in a tiny corner of the universe where only 70 had been seen before. The images, taken by MeerKAT telescope, are an indication of the detail the southern hemisphere's most powerful radio telescope may be able to provide when it is fully operational later this year. "Based on the results being shown today, we are confident that after all 64 dishes are in place, MeerKAT will be the world's leading telescope of its kind until the advent of SKA," Professor Justin Jonas, SKA South Africa chief technologist, said in a statement Square Kilometer Array The SKA, intended to be operational by the 2020s, will consist of around 3,000 dishes spread across a one square kilometer (0.4 square mile) area and will allow astronomers to peer deeper into space than ever before. SKA says it will have a discovery potential 10,000 times that of the most advanced modern instruments and will explore black holes, supernovae, dark energy and look into the origins of the universe. More than 20 countries are members of SKA, with Australia and South Africa being the main bases of operation. The project is headquartered in the UK. 'Exceptionally beautiful images' MeerKAT's images, taken of a patch of sky covering less than 0.01% of the total, reveal more than 1,300 galaxies in the distant universe, where only around 70 had been previously detected. In this image taken by the MeerKAT radio telescope, we see a galaxy approximately 200 million light years away where hydrogen gas is being used up to form stars in large numbers. They include a galaxy around 200 million light years away where new stars are being formed from hydrogen gas in large numbers, and a massive black hole spewing out jets of powerful electrons moving at close to the speed of light. A "Fanaroff-Riley Class 2" (FR2) object: a massive black hole in the distant universe (matter falling into it produces the bright dot at the center) launching jets of powerful electrons moving at close to the speed of light. "Today's exceptionally beautiful images... demonstrate that MeerKAT has joined the big leagues of world radio astronomy," said Fernando Camilo, SKA South Africa chief scientist. Want to know more? MeerKAT science and specifications Find out more about the technical specifications for the MeerKAT array, antennae and receivers. MeerKAT factsheet Download the latest MeerKAT factsheet. MeerKAT photographs Download a selection of MeerKAT visuals and photographs. MeerKAT The South African MeerKAT radio telescope, currently being built some 90 km outside the small Northern Cape town of Carnarvon, is a precursor to the Square Kilometre Array (SKA) telescope and will be integrated into the mid-frequency component of SKA Phase 1. The SKA Project is an international enterprise to build the largest and most sensitive radio telescope in the world, and will be located in Africa and Australia. Who is manufacturing the MeerKAT antennas Stratosat Datacom (Pty) Ltd, the primary industry partner on the manufacturing of the MeerKAT antennas, leads a technology consortium including international partners General Dynamics Satcom (GDSatcom, USA) and Vertex Antennentechnik (Germany). At least 75% of the components making up the MeerKAT dish will be manufactured in South Africa by several sub-contractors. Key local suppliers include Efficient Engineering (pedestals and yokes); Titanus Slew Rings (azimuth bearings) and Tricom Structures (back-up structure), all based in Gauteng. In some cases foreign companies will manufacture components for the first antenna - such as the first set of reflector panels, first receiver indexer and first sub-reflector - but the rest will be made locally. Suppliers from abroad include the National Research Council of Canada, through the Herzberg, Programs in Astronomy and Astrophysics (low-noise amplifiers and first sub-reflector); Oxford Cryosystems Ltd. (cryogenic coldheads) and Vertex Antennentechnik (control systems). MeerKAT Schedule Array Release 1 (6 receptors) Engineering Verification completed by 5 April 2016 16 receptors and correlator available Array Release 1 Science Commissioning completed 31 May 2016 once 6 done, the additional antennas gets added on a rolling basis (i.e. array commissioning significantly simplified) 16 Antenna Array Science capable by 30 June 2016 (Commissioning done and science capability, but science on PI projects not scheduled to start by then) Array Release 2 (32 receptors) Engineering Verification completed Dec 2016 (additional single receptors Engineering Verification completed) Array Release 2 (32 receptors) Science commissioning completed 31 March 2017 Early Science (PI projects) starts WB Imaging Pulsar Timing additional science modes to follow Array Release 3 (64 antennas) Engineering Verification completed mid 2017 Array Release 3 (64 antennas) Science Commissioning completed in 2017 WB Imaging Pulsar Timing additional science modes to follow MeerKAT's make-up The MeerKAT telescope will be an array of 64 interlinked receptors (a receptor is the complete antenna structure, with the main reflector, sub-reflector and all receivers, digitisers and other electronics installed). The configuration (placement) of the receptors is determined by the science objectives of the telescope. 48 of the receptors are concentrated in the core area which is approximately 1 km in diameter. The longest distance between any two receptors (the so-called maximum baseline) is 8 km. Each MeerKAT receptor consists of three main components: The antenna positioner, which is a steerable dish on a pedestal; A set of radio receivers; A set of associated digitisers. The antenna positioner is made up of the 13.5 m effective diameter main reflector, and a 3.8 m diameter sub-reflector. In this design, referred to as an 'Offset Gregorian' optical layout, there are no struts in the way to block or interrupt incoming electromagnetic signals. This ensures excellent optical performance, sensitivity and imaging quality, as well as good rejection of unwanted radio frequency interference from orbiting satellites and terrestrial radio transmitters. It also enables the installation of multiple receiver systems in the primary and secondary focal areas, and provides a number of other operational advantages. The combined surface accuracy of the two reflectors is extremely high with a deviation from the ideal shape being no more than 0.6 mm RMS (root mean square). The main reflector surface is made up of 40 aluminium panels mounted on a steel support framework. This framework is mounted on top of a yoke, which is in turn mounted on top of a pedestal. The combined height of the pedestal and yoke is just over 8 m. The height of the total structure is 19.5 m, and it weighs 42 tons. The pedestal houses the antenna's pointing control system. Mounted at the top of the pedestal, beneath the yoke, are an azimuth drive and a geared azimuth bearing, which allow the main and sub-reflectors, together with the receiver indexer, to be rotated horizontally. The yoke houses the azimuth wrap, which guides all the cables when the antenna is rotated, and prevents them from becoming entangled or damaged. The structure allows an observation elevation range from 15 to 88 degrees, and an azimuth range from -185 degrees to +275 degrees, where north is at zero degrees. The steerable antenna positioner can point the main reflector very accurately, to within 5 arcseconds (1.4 thousandths of a degree) under low-wind and night-time observing conditions, and to within 25 arcseconds (7 thousandths of a degree) during normal operational conditions. About MeerKAT - how it works Electromagnetic waves from cosmic radio sources bounce off the main reflector, then off the sub-reflector, and are then focused in the feed horn, which is part of the receiver. Each receptor can accommodate up to four receivers and digitisers mounted on the receiver indexer. The indexer is a rotating support structure that allows the appropriate receiver to be automatically moved into the antenna focus position, depending on the desired observation frequency. The main function of the receiver is to capture the electromagnetic radiation and convert it to an voltage signal that is then amplified by cryogenic receivers that add very little noise to the signal. The first two receivers will be the L-Band and UHFBand Receivers. Four digitisers will be mounted on the receiver indexer, close to the associated receivers. The function of the four digitisers is to convert the radio frequency (RF) voltage signal from the receiver into digital signals. This conversion is done by using an electronic component called an analogue to digital converter (ADC). The L-band digitiser samples at a rate of 1 712 million samples every second. (The amount of data that is generated by the digitiser for a receiver is equivalent to approximately 73 000 DVDs every day or almost 1 DVD per sec.) Once the signal is converted to digital data, the digitiser sends this data via buried fibre optic cables to the correlator, which is situated inside the Karoo Array Processor Building (KAPB) at the Losberg site complex. A total of 170 km of buried fi bre cables connect the receptors to the KAPB, with the maximum length between the KAPB and a single antenna being 12 km. Thefi bre cables run inside conduits buried 1 m below the ground for thermal stability. At the KAPB, the signals undergo various stages of digital processing, such as correlation - which combines all the signals from all the receptors to form an image of the area of the sky to which the antennas are pointing - and beam-forming, which coherently adds the signals from all the receptors to form a number of narrow, high sensitivity beams used for pulsar science. The science data products are also archived at the KAPB with a portion of the science archive data moved off site via fi bre connection and stored in Cape Town (with possibilities of reprocessing the data). Time and frequency reference signals are distributed, via buried optical fi bres, to every digitiser on every receptor, so that they are all synchronised to the same clock. This is important to properly align the signals from all receptors. The control and monitoring system is responsible for monitoring the health of the telescope and for controlling it to do what the operators want it to do. A large number of internal sensors (more than 150 000) monitor everything from electronic component temperatures to weather conditions and power consumption. Why MeerKAT? The telescope was originally known as the Karoo Array Telescope (KAT) that would consist of 20 receptors. When the South African government increased the budget to allow the building of 64 receptors, the team re-named it "MeerKAT" - ie "more of KAT". The MeerKAT (scientific name Suricata suricatta) is also a muchbeloved small mammal that lives in the Karoo region. The KAT-7 precursor array has been constructed and is being used as an engineering and science prototype. Over the weekend, the first images from the MeerKAT super radio telescope were released, showing 1,300 galaxies in a tiny spot of the universe that was previously only known to contain 70 – meaning at least 1,230 had remained hidden until now. While impressive already, the images taken this weekend are less than a quarter of what will be possible using telescope when it's fully up and running, expected to be by the mid-2020s. The images used just 16 dishes commissioned at the same time (making it still the most powerful radio telescope going) but is set to reach its full complement of 64 next year when integrated into the Square Kilometre Array (SKA). ADVERTISEMENT This setup will be able to collect data from an area of more than 1,000,000 square metres by having nodes spread across the globe, focused around Africa and Australia. More than 20 countries are taking part in the project, and the headquarters for the project is in the UK. ADVERTISEMENT Although one image of space might look a lot like another image of space to the untrained eye, the ability to take snaps of galaxies hundreds of millions of years away holds huge potential for our understanding of space. To get the project to this point has taken more than 200 specialists, from scientists and engineers to technicians, working on bespoke hardware and software. The power of the array is clearly not being underestimated, however, as more than 500 groups already have requests lodged to make use of it sometime between next year and 2020. Part of the ensemble of dishes forming South Africa's MeerKAT radio telescope is seen in Carnarvon on July 16, 2016 Even operating at a quarter of its eventual capacity, South Africa's MeerKAT radio telescope showed off its phenomenal power Saturday, revealing 1,300 galaxies in a tiny corner of the universe where only 70 were known before. The image released Saturday was the first from MeerKAT, where 16 dishes were formally commissioned the same day. MeerKAT's full contingent of 64 receptors will be integrated next year into a multi-nation Square Kilometre Array (SKA) which is is set to become the world's most powerful radio telescope. The images produced by MeerKAT "are far better that we could have expected," the chief scientist of the SKA in South Africa, Fernando Camilo said at the site of the dishes near the small town of Carnarvon, 600 kilometres north of Cape Town. This "means that this telescope as is today, only one quarter of the way down (to its full contingent) is already the best radio telescope in the southern hemisphere," Camilo told AFP. When fully up and running in the 2020s, the SKA will comprise a forest of 3,000 dishes spread over an area of a square kilometre (0.4 square miles) across remote terrain around several countries to allow astronomers to peer deeper into space in unparallelled detail. It will have a discovery potential 10,000 times greater than the most advanced modern instruments and will explore exploding stars, black holes, dark energy and traces of the universe's origins some 14 billion years ago. MeerKAT First Light image. Each white dot represents the intensity of radio waves recorded with 16 dishes of the MeerKAT telescope in the Karoo (when completed, MeerKAT will consist of 64 dishes and associated systems). More than 1300 individual objects - galaxies in the distant universe - are seen in this image. Credit: MeerKAT MeerKAT is being built in the remote and arid southwest of the Karoo region of South Africa that offers prime conditions for astronomers. It will serve as one of the two main clusters of SKA. The other will be in Australia. Montage of MeerKAT First Light radio image and four zoomed-in insets. The two panels to the right show distant galaxies with massive black holes at their centers. At lower left is a galaxy approximately 200 million light years away, where hydrogen gas is being used up to form stars in large numbers. Credit: MeerKAT Some 200 scientists, engineers and technicians working in collaboration with industry, local and foreign universities have developed the technologies, hardware and software systems for MeerKAT. 'Profoundly powerful instrument' South African Minister of Science and Technology Naledi Pandor told AFP that "this the first time that an African group of countries will host global science infrastructure of this character." Part of the ensemble of dishes forming South Africa's MeerKAT radio telescope is seen in Carnarvon on July 16, 2016 "It's a first for us as Africa and also it's a first for the world because the world hasn't done this in Africa," said the Minister. "We are building a global infrastructure for the world." "We can now expect when the 64 dishes are in place next year, it will be the best telescope, not only in the southern hemisphere but in the world," said Pandor. More than 20 countries are members of the SKA, including Britain which hosts the headquarters of the project. Despite its slowing economy, South Africa, which hosts the bulk of the SKA project, has so far invested three billion rands ($205 million) into the telescope project, funded mainly from the public purse and science research partners. When fully up and running in the 2020s, the SKA will comprise a forest of 3,000 dishes spread over an area of a square kilometre (0.4 square miles) across remote terrain around several countries Already some 500 scientific groups from 45 countries have booked slots to use the MeerKAT array between next year and 2022. "What this will do is bring to South African and world astronomers, the most astonishing and profoundly powerful instrument ever used before in radio astronomy," SKA South Africa project director Rob Adam told AFP. Explore further: First antenna launched on precursor to world's largest telescope | Summary: Scientists promised it would be the world's most powerful radio telescope, capable of exploring dark energy, black holes, and traces of the universe's creation 14 billion years ago. South Africa's MeerKAT has already exceeded expectations. Though operating at a quarter of its eventual capacity with 16 of 64 planned dishes, MeerKAT's first image, released Saturday, revealed some 1,300 unknown galaxies in a tiny patch of sky previously known to house only 70, reports AFP. Its other "exceptionally beautiful" images "are far better (than) we could have expected," says Fernando Camilo of Square Kilometer Array-a multi-radio telescope project involving 200 specialists and 20 countries that will eventually include 3,000 dishes, mainly in South Africa and Australia, reports Wired. The images feature a galaxy forming new stars from hydrogen gas and a black hole emitting electrons close to the speed of light, reports CNN. Naledi Pandor, South Africa's science minister, tells IOL that the quality of images is what scientists expected from 32 dishes, not 16, meaning MeerKAT "is already the best radio telescope in the southern hemisphere," says Camilo. "When the 64 dishes are in place next year, it will be the best telescope, not only in the southern hemisphere but in the world," adds Pandor. When it hits full capacity in the 2020s, SKA "will have a discovery potential 10,000 times greater than the most advanced modern instruments," reports AFP. Wired reports some 500 groups have already asked to book MeerKAT between 2017 and 2020. | 5,157 | 378 | multi_news | en |
Summarize: CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of my prior co-pending application Ser. No. 124,813 filed: Feb. 26, 1980, now abandoned. BACKGROUND OF THE INVENTION The present invention relates to water distributing apparatus and more particularly to a water sprinkler system for use in uniformly wetting a roof structure. References known to the present applicant and believed to be relevant to the present invention include the following U.S. Pat. Nos. 1,831,880 issued to Pierce; 2,865,674 issued to Jelmeland; 3,587,972 issued to Weeth; and 3,633,826 issued to Baker. The Pierce patent teaches a fire protection and roof cooling arrangement comprising basically a length of perforated pipe positioned on the ridge of a roof. The Jelmeland patent also teaches a roof cooling and fire prevention system in which somewhat conventional spray nozzles are positioned along the eaves of a roof to provide a water spray reaching all the way to the roof ridge. This patent also recognizes that in general excess amounts of water must be sprayed onto a roof to fully wet it with the result that much water runs off. The Jelmeland patent includes a recirculating pump system to avoid wasting the runoff water. The patents to Weeth and Baker each provide apparatus for distribution of water in irrigation or lawn sprinkling systems. In each of these patents, a generally vertically extending flexible tube or hose is used as a discharge nozzle from a main water conduit to distribute irrigation water over as large a ground surface as possible. The normal reaction force produced by water flowing from the nozzle causes the flexible tube or hose in each of these patents to rotate in a generally circular pattern with the end of the hose directed at a fairly high angle, on the order of 45°, to provide maximum distribution of the water. In the Baker patent, it is also taught that splitting of the end of the rubber tube will break up the flow of water from the nozzle into a spray of small water droplets more desirable in the lawn sprinkling situation. As taught by the first two above referenced patents, the sprinkling of water on a building roof will aid in cooling the entire structure. As can also be seen from those patents, the previously known systems tend to distribute much more water onto the roof structure than is actually needed for proper wetting. The perforated pipe arrangement taught by Pierce tends to produce a large number of distinct rivulets of water extending from each pipe perforation to the roof edge with dry roof areas in between. The sprinkler arrangement of Jelmeland breaks the water up into small droplets which are randomly distributed and therefore can provide essentially complete wetting of the roof. However, such a sprinkler arrangement is inefficient, especially if windy conditions are considered. The water droplets which must travel a great distance through the air before striking the roof may partly evaporate while in the air and tend to be carried completely away from the roof depending on the ambient wind velocity. As illustrated by the drawings in that patent, a considerable amount of the spray will extend beyond the roof edges to insure that the entire roof structure is wet. Excess water is also applied to be sure of complete wetting and then the excess is collected and recirculated. Another problem found in these prior systems is that relatively high water supply pressure must be provided to the sprinkler head. For example, in the Jelmeland device, pressure must be sufficient to break the water into a fine mist and propel the mist to the roof ridge. In the Weeth and Baker devices the flexible tube is relatively stiff, and relatively high water pressure is required to cause the desired rotation. It can be seen, therefore, that it is desirable to provide a system for wetting a roof structure to aid in cooling the structure, thereby reducing the cost of air conditioning the structure in warm climates. It can also be seen that it is desirable to provide uniform wetting of a roof structure and to do so using the least amount of water and at relatively low pressure. While it is desirable to uniformly distribute water on the roof structure, it can be seen that it is not desirable to break the water up into small droplets and spray them over long distances to the roof surface since windy conditions can cause considerable loss of water when such arrangment is employed. SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide an improved water distributing system for wetting a structure roof. Another object of the present invention is to provide a roof sprinkling system which provides uniform roof wetting with minimum water usage. Another object of the present invention is to provide a roof sprinkling system which provides uniform roof wetting at a relatively low supply pressure. Another object of the present invention is to provide a roof sprinkling system which provides minimum exposure of the distributed water to wind as it is applied to the roof surface. Yet another object of the present invention is the provision of a roof sprinkling system which is reliable in operation, requires minimum maintenance and is easily assembled. A roof sprinkling system according to the present invention comprises a water supply conduit adapted for attachment along the ridge of a roof, a flexible tube having a first end attached to the top of the water supply conduit, and a directional nozzle attached to a second end of the flexible tube with said nozzle having an outlet angularly displaced from the longitudinal axis of the second end of the flexible tube. In one preferred form, the flexible tube first end is attached to the supply conduit by a fitting including a pressure reducing orifice. In another preferred form of the present invention, the supply conduit includes spaced apart conduit tee fittings having an upstanding branch conduit portion including a plug in which the tube fitting is disposed. A tubular sleeve projects upward beyond the point of attachment of the flexible tube at its first end for engagement of the tube to control the flailing action thereof. A filter screen may be provided in the plug to prevent clogging of the pressure reducing orifice and directional nozzle. BRIEF DESCRIPTION OF THE DRAWINGS The present invention may be better understood by reading the following detailed description of the preferred embodiments with reference to the accompanying drawings wherein: FIG. 1 is an illustration of a roof sprinkling system according to the present invention installed on a building structure; FIG. 2 is a perspective illustration of a portion of the water sprinkling system of FIG. 1; FIG. 3 is a cross-sectional illustration of a portion of the FIG. 2 apparatus; FIG. 4 is a detailed illustration of the nozzle portion of the sprinkling apparatus of FIGS. 1 and 2. FIG. 5 is an illustration of a portion of a roof sprinkling system in accordance with another embodiment of the present invention; and FIG. 6 is a vertical longitudinal section view of a portion of the system shown in FIG. 5. DESCRIPTION OF THE PREFERRED EMBODIMENTS With reference now to FIG. 1, there is provided a simplified illustration of a water distributing system, generally designated 10, according to the present invention installed on a roof 12 of a building structure 14. The water sprinkling apparatus 10 comprises primarily a length of water supply conduit 16 attached to the roof 12 and a large number of flexible discharge tubes 18 attached to the upper surface of conduit 16. The water supply conduit 16 is, in the preferred embodiment, a length of nominal three-quarter inch PVC plastic pipe. The pipe 16 is preferably bolted or otherwise attached using suitable brackets to the roof 12 at or near the ridge 20. As illustrated in FIG. 1, the conduit 16 may be positioned slightly below ridge 20 so that the water sprinkler system 10 cannot be seen from the front of the building 14. The details of the flexible discharge tubes 18 and their attachment to conduit 16 are shown in FIGS. 2, 3 and 4. The discharge tubes 18 in this preferred embodiment are provided every 2.5 feet along the length of the supply conduit 16. Water may be supplied to the conduit 16 in a number of ways. In this preferred embodiment, additional piping 22 is provided from the conduit 16 to a point on an outer wall of the structure 14. The permanent conduit 22 may be connected to a conventional water tap 24 by means of a flexible hose 26 to allow draining of the system during freezing weather as well as for other possible reasons. In this preferred embodiment, the flow of water into the sprinkler system is controlled by an electrically operated valve 28 which is in turn controlled by a clock mechanism 30. The electrical timing system 30 and valve 28 may be the same as those commonly employed on automatic yard sprinkler systems. Such systems can automatically open the valve 28 for preselected time periods and at desired intervals throughout the day. The precise setting of the control 30 depends on the particular dimensions of the roof to be cooled, the dimensions of the sprinkler system itself and the available water pressure. In general, the sprinkler time period should not be so long as to allow excess water to run off the eaves of the roof 12. If desired, the roof 12 may be provided with guttering 32 along its eaves to catch the excess runoff water. As shown in FIG. 1, a lawn sprinkler hose 34 may be attached to downspout 36 so that any runoff water may be distributed to desired points on the surrounding lawn to provide maximum utilization of the water. With reference now to FIG. 2, more details of the water conduit 16 and discharge tubes 18 are provided. As noted above, the conduit 16 is preferably PVC plastic pipe which is basically rigid. The water discharge tubes 18, on the other hand, are very flexible plastic material, having an outer diameter of one-eighth inch and an inner diameter of one-sixteenth inch. The flexible tubes 18 are preferably attached to the rigid conduit 16 by brass fittings 38 which are threaded into holes which have been drilled and tapped into the upper surface of conduit 16. Fittings 38 have a barbed upper end onto which the flexible tubes 18 are frictionally pressed to provide essentially permanent engagement. The details of the fittings 38 are provided in FIG. 3 and described in more detail below. The flexible tubes 18 are generally between six and seven inches long and carry directional nozzles 40 in their free ends. Each of the nozzles 40 comprises a substantially rigid tube bent at about its midpoint and having one end frictionally pressed into the flexible tubes 18. More details of the nozzles 40 are illustrated in FIG. 4 and described below. With reference now to FIG. 3, details of the attachments of the first ends of tubes 18 to the conduit 16 are provided in a cross-sectional illustration. As noted above, a threaded hole 42 is provided in the upper surface of conduit 16 at the location of each flexible discharge tube 18. The fitting 38 includes a threaded lower end 44 which engages the threaded hole 42. A rubber washer 46 provides a water tight connection between the fitting 38 and the conduit 26. A large diameter portion 48 of the fitting 38 is hexagonally shaped so that a wrench may be used for tightening fitting 38 into conduit 16. An upper section 50 of fitting 38 is barbed to provide a water tight connection of the tube 18 to fitting 38 and to resist removal of the tubing 18. Fitting 38 has an axial passageway 52 providing communication between conduit 16 and flexible tube 18. In a preferred form, the upper portion of passageway 52 has an inner diameter of 3/64 inch over a length of about 3/16 inch and acts as a pressure-reducing orifice as explained in more detail below. With reference now to FIG. 4, details of the water discharge nozzle 40 are illustrated. In the preferred embodiment, nozzle 40 is formed from a one-inch length of brass tubing having an inner diameter of 1/16 inch and an outer diameter of slightly greater than 1/8 inch. The nozzle 40 is bent at its midpoint 42 to an angle indicated by the arrow 44 of approximately 30°. As shown in FIG. 4, this angle is measured relative to the longitudinal axis of that portion of nozzle 40 which is inserted in the second end of flexible tube 18. As a result, the angle 44 also represents the angular displacement of the nozzle 40 outlet 46 from the longitudinal axis of the second end of the flexible tube 18 itself. With reference now to all of the figures, the operation of the present invention will be described. With no water pressure supplied to the system, the flexible tubes 18 tend to be bent over so that nozzles 40 are aiming in a direction well below horizontal and are generally at the same elevation as the fitting 38. When water pressure is applied to the system, for example by the actuation of valve 28, the flexible tubes 18 experience several different forces. Internal pressure within the tubes 18 tends to cause these tubes to stand more erect. The small diameter orifices provided in passageways 52 in fittings 38 help reduce the average pressure within the flexible tubes 18 to thereby aid in preventing the tubes 18 from standing at too high an angle. These fluid-reducing orifices also aid in equalizing distribution of fluid flow through the large number of flexible tubes 18 on the roof ridge. The reaction force of water exiting nozzles 40 also tends to push the free ends of tubes 18 upwards. The brass discharge nozzles 40 themselves add weight to the free ends to help overcome this lifting reaction force. It can be seen that if the tubes were allowed to stand erect as in the prior art flexible tube sprinkler systems, a high angle water spray would result which would be subject to being carried off the roof 12 by wind. In the present invention, the very high ratio of length to diameter of tube 18 combined with the weight of nozzles 40 helps to insure that the water discharged from nozzles 40 travels in a generally horizontal direction as it leaves the nozzles. It can be seen that the water leaving nozzles 40 forms a stream of large droplets which is less subject to wind dispersion than a fine mist would be. It is desirable that the stream be rapidly and randomly directed over as large a portion of the roof as possible. The bend or offset in the discharge nozzles 40 guarantees the random motion of the flexible tubes 18. Thus, the reaction force of water leaving the outlet 46 of the discharge nozzle 40 includes a component which is perpendicular to the longitudinal axis of the free end of flexible tube 18. As a result, the free end of tube 18 is always displaced sideways relative to whatever position it happens to be in. In the prior art flexible tube sprinklers, it is noted that the free end of flexible tube tends to travel in a precise circle to provide even distribution of the water. It can be seen that if the free ends of flexible tubes 18 traveled in an exact circle, the nozzles 40 would experience a rotation about the longitudinal axis of the tube 18 free ends. As a result, the direction of the sideways force on the free ends of tubes 18 would vary as the tube rotated in the circular path. That is, at some points in the rotation, the sideways force would be upwards while in others it would be downwards. As a result of this offset in the outlet of nozzles 40, the free ends of tubes 18 do not travel in an exact circular arc, but instead travel in a very rapid and random fashion, distributing water over a fairly large area considering the small size of the tubes 18. While providing this large distribution, the fluid distributing system does not spray the water upwardly to any great extent, thus minimizing the loss of water due to wind. In testing the present invention under high wind conditions, a second beneficial effect of the very great length to diameter ratio of tubes 18 was discovered. At high wind conditions, the force applied to the tubes 18 by the wind tends to make the free ends and therefore the nozzles 40 extend farther from the fittings 38 and to thereby travel closer to the surface of roof 12 and to discharge water at an even lower angle. Thus, the apparatus of the present invention not only avoids loss of water due to the wind because of its low angle of discharge, but actually provides an even lower angle of discharge under high wind conditions to further minimize losses. Another preferred embodiment according to the present invention is illustrated in FIGS. 5 and 6 of the drawings. Referring to FIGS. 5 and 6 a water sprinkler apparatus according to the present invention is illustrated, in part, disposed on the roof 12 in a manner similar to the apparatus illustrated in FIG. 1. The water sprinkler apparatus according to the embodiment shown in FIG. 5 comprises a main water supply conduit characterized by sections of pipe 60 interconnected with spaced apart tee fittings 62, as shown, to form an elongated conduit means at or near the ridge 20. The conduit formed by the pipe sections 60 and tee fittings 62 may be connected to the permanent conduit 22 in a manner similar to the arrangement shown in FIG. 1. If a tee fitting is disposed at the end of the main supply conduit, it may be provided with a suitable plug 63 in place of a continuing section of conduit 60. The conduit sections 60 and the tee fittings 62 are also preferably formed of PVC plastic pipe. The conduit assembly formed by the conduit sections 60 and tee fittings 62 may be suitably retained on the roof 12 by spaced apart brackets 64, for example. As shown in FIG. 5, the spaced apart tee fittings 62 are arranged to have their respective base leg portions 66 projecting substantially vertically upward and forming branch conduits to which are suitably connected the flexible tubes 18, as shown in FIGS. 5 and 6. Referring particularly to FIG. 6 one of the tee fittings 62 is shown in longitudinal section. The tee fittings 62 are each provided with a plug 68 insertable in the upstanding base leg portion 66. The plug 68 includes a transverse end wall 69 provided with a tapped hole 70 for receiving the fitting 38 threadedly engaged therein. The tee fitting 62 is also provided with filter means comprising a fine mesh screen 72 disposed between the bottom of the plug 68 and a supporting collar 74 insertable within the bore of the base leg 66 and suitably retained therein by a locating ridge or shoulder 76 formed in the tee fitting. The screen 72 provides for filtering out particulate matter entrained in the water to prevent clogging the passageway 52 within the fitting 38, which acts as a pressure reducing orifice, and also to prevent clogging of the directional nozzle 40. The tee fitting 62 and the plug 68 may be formed with cooperating threads, as shown, so that the plug can be removed periodically for cleaning the filter screen 72 as needed. An important aspect of the embodiment of the present invention shown in FIGS. 5 and 6 pertains to the provision of an upstanding tubular sleeve 78 disposed around the base leg 66 of the tee fitting and suitably secured thereto by an adhesive or interference fit. The tubular sleeve 78 projects upwardly in surrounding relationship to the fitting 38 and the end of the tube 18 connected to the fitting, and is delimited by an upper edge 80. It has been determined that the provision of the tubular sleeves 78 improves the circular swinging or flailing action of the tubes 18 to provide a more uniform motion of the tubes and also provide some support for the tubes near the point of connection to the respective fittings 38. The provision of the tubular sleeves 78 also reduces the tendency for the nozzle 40 to be snagged by the edges of the roof shingles, for example, by supporting tube 18 so that the nozzle 40 does not lay against the roof surface when at rest, or in motion, as a result of water or wind forces. The sleeves 78 are provided with diametrally opposed openings 81 providing drains for any water which might collect within the sleeve and around the plug 68. In a preferred embodiment of the water sprinkler apparatus illustrated in FIGS. 5 and 6 the conduit sections 60 and tee fittings 62 are conventional PVC plastic pipe fittings of nominal diameter of one and three quarters inches. The plug 68 and the sleeve 78 may also be formed of PVC or other suitable plastic materials. The filter 72 is preferably formed of a copper mesh screen. Various modifications within the scope of the present invention will be apparent to those skilled in the art. For example, the fittings 38 may be formed as separate plastic parts glued into holes drilled into the conduit 60 or the plugs 68, or the fittings could conceivably be molded in place in the respective members. In similar fashion, the nozzles 40 may be formed of materials other than brass, although it is generally believed desirable to employ a material having greater weight per unit length than the tubes 18 to aid in lowering the free ends of the tubes. It is also apparent that the dimensions of the various parts of the apparatus disclosed herein may be modified to increase or decrease the total water flow rate and to compensate for variations in available water pressure. The systems taught herein have been found to provide for effective water distribution at pressures as low as ten pounds per square inch due to the rapid random flailing motion induced by the offset outlet arrangement of the nozzles 40. | Summary: Water distributing apparatus for uniformly wetting a roof to provide evaporative cooling. The apparatus includes a generally horizontal water conduit supported on a roof, a flexible tube attached to an upper surface of the conduit and a directional nozzle attached to a second end of the flexible tube. The directional nozzle has an outlet angularly displaced from the longitudinal axis of the second end of the flexible tube to provide rapid generally random movement of the nozzle about its point of attachment to the water supply conduit. In one preferred embodiment the conduit includes spaced apart conduit tee components having upstanding branches into which are fitted plugs supporting connector fittings for the flexible tubes. A filter screen is mounted in each of the plugs. An upstanding sleeve surrounds the tube connector fitting and is operable to engage the flexible tube to minimize snagging of the directional nozzle under the edges of the roof shingles and control the flailing action of the tubes. | 5,038 | 200 | big_patent | en |
Write a title and summarize: Cutaneous leishmaniasis (CL), caused by Leishmania braziliensis, is the most important presentation of tegumentary leishmaniasis (TL) in Latin American. While the role of dogs as reservoirs of Leishmania infantum, and the clinic features of canine visceral leishmanisis are well described, little is known about the importance of dogs in the transmission of L. braziliensis to humans. In the present study, we determine the frequency of L. braziliensis infection in dogs with cutaneous and mucosal ulcers in an endemic area of CL. We also describe the clinical manifestations and histopathologic features, and determine if the parasites isolated from dogs are genetically similar to those found in humans. This is a cross sectional study in which 61 dogs living in an endemic area of CL and presenting ulcerated lesions were evaluated. Detection of L. braziliensis DNA by polymerase chain reaction (PCR) in skin biopsies, serology and leishmania skin test (LST) with soluble L. braziliensis antigen were performed. The clinical and histopathologic features were described, and we compared the genotypic characteristics of isolates obtained from dogs and humans. The sensitivity of the three tests together to detect exposure was 89% and the concordance between the tests was high. The skin lesions were most frequent in the ears, followed by scrotal sac. The PCR was positive in 41 (67%) of animals, and the lesions in the snout, followed by the scrotal sac and ears were the sites where parasite DNA was most detected. There were genotype similarities between L. braziliensis isolates from dogs and humans. The high frequency of L. braziliensis infection in dogs with ulcers and the similarities between the isolates of L. braziliensis and cutaneous leishmaniasis in dogs and humans in an endemic area of TL, raise the possibility of an important role of dogs in the transmission chain of L. braziliensis. Leishmaniasis is an antropozoonosis caused by the protozoa of the genus Leishmania, which is transmitted to humans by infected sandflies. Leishmaniasis is classified as visceral leishmaniasis (VL) and tegumentary leishmaniasis (TL). Approximately 20 Leishmania species may cause human disease. In the New World, VL is caused by Leishmania infantum and TL is mainly caused by Leishmania Viannia braziliensis, Leishmania Viannia guaynensis and Leishmania mexicana mexicana [1]. Foxes and the dogs are the most common wild and urban reservoir for L. infantum, and play a key role in the persistence of the disease [2]. Unlike VL, very little is known about the wild and urban reservoirs of parasites that cause TL in the New World. L. braziliensis is the most important causal agent of leishmaniasis in Latin America. Cutaneous leishmaniasis (CL) is the most frequent form of TL, occurring in more than 90% of patients. Additionally, about 3% of patients infected with L. braziliensis develop mucosal leishmaniasis and 2% disseminated leishmaniasis [3–5]. Amastigote forms of Leishmania have been documented in tissue from dogs, donkeys, horses and rats in endemic areas of L. braziliensis, raising the possibility of the role played by these animals in L. braziliensis transmission [6,7]. In endemic areas of CL the exposure of dog to Leishmania sp have been documented by serologic tests, identification of amastigotes in biopsies of cutaneous lesions, or isolation of parasite in aspirated material from skin cutaneous ulcers [8]. However, the identification of the species of Leishmania was performed in only a few reports and there is a lack of studies describing the clinical and histopathologic features of the L. braziliensis infection in dogs. Unlike canine VL, a disease that is well characterized by different clinical forms, including asymptomatic, oligosymptomatic, and symptomatic clinic canine VL [9], there is no clinical and histopathologic characterization of TL in dogs. Currently, the most studies included just a small number of animals evaluated and without identification of the parasite species [6,10]. Recently in the southern region of the state of Bahia, Brazil, a known endemic area for L. braziliensis, 560 dogs were analyzed for the presence of skin lesions of CL. Anti-Leishmania antibodies and a polymerase chain reaction (PCR) for detection of L. braziliensis DNA were performed. Specific primers were used to differentiate between L. braziliensis [11] and L. infantum [12]. In this study, 32 (6%) of the 560 dogs presented lesions suggestive of CL, and the PCR for L. braziliensis was positive in 54. 7% of 413 skin biopsies obtained from dogs [13]. Genetic differences from isolates of Leishmania belonging to the same species have been widely documented [14–16]. Moreover, L. braziliensis is polymorphic and genotypic differences are associated with different clinical forms of the disease [17,18]. Thus, it is important in epidemiologic studies to determine if the species found in humans are similar to those found in animals, which may have the potential to participate in the transmission chain. Additionally, polymorphisms of L. braziliensis isolates from dogs have been detected [19]. However, no studies have been performed to evaluate if isolates of L. braziliensis from dogs are similar to the parasites isolated from humans. These data clearly indicate the need for studies that characterize canine cutaneous leishmaniasis and the role of dogs as a reservoir of L. braziliensis. The aim of the present study was to determine the frequency of L. braziliensis infection in dogs with cutaneous and mucosal ulcers in an endemic area of human cutaneous leishmaniasis. Further, the study aimed to describe the clinical manifestations and the histopathology features of canine CL caused by L. braziliensis, and to determine if the parasites isolated from dogs are genetically similar to those found in humans. This study was performed in strict accordance with the recommendations of Brazilian Federal Law on Animal Experimentation. The protocol was approved by the Ethics Committee for the Use of Animals in Research (CEUA license number: 015/2017) from the Gonçalo Moniz institute (IGM-FIOCRUZ). The owners of the dogs gave written informed consent prior to sample collection and the skin sampling procedures were performed under sedation. No adverse events were recorded during the sample collection. The study was conducted in the village of Corte de Pedra, Bahia, Brazil, where TL due to L. braziliensis is endemic. The village is characterized by isolated sites of secondary forest, with agricultural activities providing the main source of income for the majority of the population. Agricultural work increases exposure to L. braziliensis through increased contact with Lutzomyia (Nyssomyia) whitmani and Lutzomyia (Nyssomyia) intermedia sandflies, which are the vectors of L. braziliensis in the region [20]. This is a cross sectional study to determine the frequency of canine leishmaniasis caused by L. braziliensis infection in dogs with ulcerated skin or mucosal lesions, to describe the clinical and histopathologic features of canine leishmaniasis caused by this species, and to determine the positivity of different diagnostic tests for L. brazilensis in these animals. Moreover, we compared genotypically the isolates of L. braziliensis from dogs with the isolates from human CL. The 50 residences closest to the medical clinic of Corte de Pedra who had dogs with ulcerated lesions participated in the study. The selection of the dogs was based on the information by mouth about the presence of dogs with ulcerated lesions, initially in the houses close to the Health Post and later in four neighborhoods close to the Heath Post. There were 61 dogs with lesions in the 50 houses. Initially, we explained to the dogs’ owners the objectives of the study and the informed consent form was read and signed by them. A questionnaire was answered about demographic characteristics of the animals, duration of the lesion and occurrence of previous or present cases of human disease in the house. The dogs were then immobilized, physical examination was performed and blood (8 mL) from the lateral saphenous vein was obtained. After the area of the lesion was cleaned with alcohol and lidocaine for anesthesia was applied, a skin biopsy of the ulcer was obtained with a punch of 4 mm and added to Eppendorf tubes containing RNA Later Solution (Ambion, Life Technologies, Thermo Fisher Scientific, USA). In animals that had more than one lesion we choose the greatest ulcer to obtain the skin biopsy. In about half of the animals, the biopsy was divided in two pieces being one added to formaldehyde and other to the RNA Later Solution. Parasites were genotyped according to the haplotypes of polymorphic nucleotides in the locus CHR28/425451, previously shown to distinguish L. (V.) braziliensis strains in Corte de Pedra [27]. Primers 5´: TAAGGTGAACAAGAAGAATC and 5´: CTGCTCGCTTGCTTTC were used to amplify a 622 nucleotide-long segment in CHR28/425451 from parasite genomic DNA as previously described [27]. Amplicons were cloned using the Original TA Cloning Kit pCR 2. 1 VECTOR (Invitrogen, Thermo Fisher Scientific Co., MA, USA), according to manufacturer’s instructions. Briefly, the amplicons were inserted by overnight ligation into PCR 2. 1 plasmids, which were used for chemical transformation of competent DH5α Escherichia coli. Plasmid minipreps were generated from four recombinant bacteria colonies per study isolate [28]. Amplicon cloning was confirmed by digestion analysis, using Eco RI restriction endonuclease (Invitrogen). Plasmid inserts were sequenced by the Sanger method with primers complementary to the M13 vector sequences. Sequencing was performed at Macrogen Inc. (Seoul, South Korea). Mega 5. 0 software [29] was used to align the sequences with the CHR28/425451 clones obtained from the panel of L. (V.) braziliensis parasites, in order to determine the SNP/indel haplotypes detectable in each study isolate. The study consisted of a panel of two groups of samples, 29 L. braziliensis isolates obtained from the dogs included in this study and 113 L. braziliensis isolates obtained from human beings in 2008–2011 periods. In order to check whether L. braziliensis strains circulating among dogs in Corte de Pedra might be shared by human CL patients in that region, the 600 base pairs long locus that starts at nucleotide position 425,451 on the parasite’s chromosome 28 (i. e. CHR28/425451) was PCR amplified, cloned, sequenced and compared across a panel of isolates of dog or human origin. For analysis of the nucleic acid sequences of the polymorphic locus CHR28/425451, the consensus sequence at the locus explored from the CL sample (i. e., 2008–2011) was first determined, which was used to compare the different L. braziliensis samples obtained from 29 animals. Then, the sequences were analyzed for the identification of the occurrence of SNPs and/or indels for the identification of polymorphism alleles. We defined polymorphism as a single difference between the sequences of the evaluated isolates and polymorphic allele as a linear DNA sequence of the locus studied, detected in more than one clone per parasite isolate, and in more than one isolate of L. braziliensis in our study sample. The frequency of distinct alleles was determined in each isolate of L. braziliensis obtained from the dog biopsy for the locus CHR28 / 425451. The alignment between the isolates from dogs and humans showing the polymorphisms is presented in S1 Fig. The distribution of variables such as age, duration of disease (years), number of lesions and site of injury were expressed by mean and standard deviation and were analyzed by the student' s T-test. The Chi-square and Fisher' s exact tests were used for comparisons of proportions between groups. The comparative analyzes of the diagnostic tests were performed by the Kappa Index of concordance. A p value of <0. 05 was considered statistically significant. The 61 dogs with skin lesions enrolled in this study lives in 50 houses. Twenty animals (33%) presented more than one cutaneous lesion and 6 (9. 8%) had also mucosal leishmaniasis. The demographic and clinical features according to the positivity of the PCR are presented in (Table 1). The percentage of males was higher in the group with positive PCR (p<0. 008) but there was no difference between age, duration of illness and number of lesions with regard to the positivity of the PCR. The site of the largest lesion in those animals with a positive PCR was in the scrotal sac, followed by the ears and in six animals the lesions were in the muzzle. The PCR was positive in the lesion tissues of 41 (67%) of the 61 animals. All the lesions were well limited ulcers with raised borders (Fig 1). The pictures are from six different animals with ulcers located in the scrotal sac (Fig 1A and 1B), in the nose (Fig 1C and 1D) and in the ears (Fig 1E and 1F). In all of these lesions, DNA of L. braziliensis was documented in the biopsies. The age of the animals presented in these pictures ranged from three years (Fig 1B) to 10 years (Fig 1C). The sensitivity of all the three tests together to detect exposure to L. braziliensis was 89%, and the concordance between the tests was high. For this analysis, we used the PCR test as the gold standard. The concordance for PCR and LST and PCR and serology was considered substantial (κ = 0. 597 and κ = 0. 674, respectively). In the 41 animals with positive PCR, antibodies were detected in 37 (90%) and the LST was positive in 32 (78%). In the 41 animals with positive serology, the LST was positive in 32 (78%). The age of the 54 dogs with at least one positive test was 6 ± 3. 2 years and among the seven animals without evidence of exposure was 3 ± 1. 9 years (p = 0. 02). The illness duration in the two groups was of 210 (90–547) and 75 (38–296) years respectively (p = 0. 12). The histopathology analysis of the cutaneous ulcer border was performed in 35 animals, (Table 2). In 26 animals (74%), amastigotes were documented, and in nine (26%) parasites were not detected. However, the histopathologic features were similar in those with or without evidence of amastigotes. In all the animals, there was a chronic inflammation with lymphocytes, plasma cells, macrophages infiltration and granulation tissue. The inflammation was not associated either with the presence of parasites, necrosis and fibrosis were documented (Fig 2). An inflammation predominantly moderate or mild was observed in 77% of the biopsies with the presence of amastigotes, and a similar finding was detected in 89% of the biopsies negative for parasites. Granuloma was observed in only three (8%) of the animals, two of which were PCR positive. Focal necrosis was observed in 42% and 55% in the biopsies with or without amastigotes, respectively. Presence of fibrosis was observed in 75% of the biopsies, and intense or moderate fibrosis was similar in animals with or without parasites. In order to check whether L. braziliensis strains circulating among dogs in Corte de Pedra might be shared by human CL patients in that region, the 600 base pairs long locus that starts at the nucleotide position 425,451 on the parasite’s chromosome 28 (i. e. CHR28/425451) was PCR amplified, cloned, sequenced and compared across a panel of isolates. After alignment of CHR28/425451 sequences, a total of 20 different alleles could be identified in parasites isolated from dogs as determined by their SNP and indel contents, while seven could be identified in L. braziliensis obtained from human beings. Overall, 24 different alleles could be enlisted, since parasites from human beings and dogs shared three distinct alleles, which were based on the contents of positions 30,286 and 545 within the locus that could be described as TT-, CCT and CC-. The frequency distribution of seven alleles in L. braziliensis locus CHR28/425451 among patients and dogs are shown on (Table 3). The frequency of isolates presenting the alleles CCT was high in both dogs and humans, and alleles TT- were present similarly in isolates of both humans and dogs. The knowledge of wild and domestic reservoirs of Leishmania are important to understanding the dynamic of the transmission of this protozoa and to the establishment of control measures, aimed to decrease transmission and consequently decrease the appearance of disease. While the role of dogs in the epidemiology of L. infantum infection is well known, little is known about the reservoirs of L. braziliensis. Here, we show that in a highly endemic area of L. braziliensis, dogs present ulcerated lesions typical of CL, and have evidences of L. brazilensis infection by documentation of DNA of L. braziliensis, documentation of parasites in histopathology examination, positive serologic test and or a positive leishmania skin test. There are previous publications calling attention to the presence of dogs presenting ulcerated lesions with evidence of Leishmania infection or even documentation of parasites in bone marrow and spleen of asymptomatics dogs in areas of L. braziliensis transmission [6,29,30]. However, the majority of these studies lack documentation that L. braziliensis was the causal agent of the infection [8,31–39]. Here, evaluating 61 dogs with ulcerated lesions, evidence of leishmaniasis infection was observed in 89%, and 67% of the animals had detection of DNA of L. braziliensis. The clinical characteristic of the lesion was a well limited ulcer with raised borders similar to the ulcers typically found in human CL due to L. braziliensis. The presence of ulcers mainly in the scrotal sac was likely due to this area being exposed to sandflies bites. The long duration of the disease in dogs differs from what has been observed in humans with L. braziliensis. The short illness duration in humans could be explained in part by the seek for medical attention and use of therapy for leishmaniasis. But, based in studies in which no therapy or use of placebos were administrated in clinical trials with human cutaneous ulcers caused by L. braziliensis, the lesions were healed within one year of illness duration [40–43]. In contrast, in this study, the majority of the dogs had illness duration for more than six months and some of them had the disease for more than three to five years. These data support the argument that dogs keep the illness for a long period of time, and the active disease may increase their ability to transmit the infection. The histopathology features of the dogs with ulcerated lesions was characterized by lymphocytes, plasma cells and macrophages infiltration as it is observed in human CL [6,26]. Nevertheless, they differ from the lesions in humans because they have less inflammation and more vascularization than human lesions. The similarities between the histopathologic findings in animals with positive or negative PCR and in dogs with or without evidence of amastigotes support the hypothesis that likely all the animals in the present study may have been infected with Leishmania. The observation that the intensity of the inflammatory reactions was moderate or light in the majority of the animals may be explained by the long illness duration of the disease and the persistence of the lesions for many years. We have previously shown that L. braziliensis in Corte de Pedra consists in a complex population made of several different strains of the parasite [18]. We further extended this understanding after cloning and comparing the sequences of several loci in different chromosomes of the L. braziliensis of Corte de Pedrda [27]. In that study, we detected six loci that presented two or more alleles in that protozoa population. In that and follow-up research, we successfully employed the 500 base-pairs locus that starts at nucleotide position 425,451 of L. braziliensis chromosome 28 to test association between parasite genotype and outcomes of leishmaniasis [27,44,45]. In the current study, we explored that same locus to determine if parasites that infect the dogs presented genotypes similar to those that infect humans. The observation that the frequency of the alleles TT- and CCT was similar in L. braziliensis isolated from dogs and humans suggests that L. braziliensis from humans and dogs share, at least in part, similar genotype profile in Corte de Pedra. We are aware that in addition to these observations others studies need to be performed to better characterize the importance of dogs and canine TL in the transmission of L. braziliensis. However, it is relevant that additionally to these findings regarding the similarities between the genotypic characteristics of humans and dogs isolates, active disease or past history of CL was documented in all houses with dogs with TL. Specifically, there were active or past history of CL in 93 individuals living in the 50 houses where canine TL was observed. This study shows that in an area of L. brazilensis transmission dogs presenting cutaneous ulcers are likely infected by L. brazilensis. The clinical and histologic features of canine CL were similar to the observed features in humans, but dogs may remain with the disease for a longer period of time. The occurrence of humans with CL or previous history of CL, and dogs with canine CL in the same house and the similarities between the parasites isolates from dogs and humans strongly argue in favor of the possibility that dogs participate in the transmission of L. braziliensis. | Title: Clinical and histopathologic features of canine tegumentary leishmaniasis and the molecular characterization of Leishmania braziliensis in dogs Summary: Cutaneous Leishmaniasis (CL) is the major clinical forms of tegumentary leishmaniasis. More than 1 million of people develop cutaneous leishmaniasis worldwide and Leishmania braziliensis is the main causal agent of the disease in Latin American. Wild and domestic animals play an important role in the maintenance of the parasites that are transmitted to humans by infected sandflies. There is no vaccine for leishmaniasis and control measures are not effective in part because it is not known the reservoirs of L. braziliensis. Here, we show that dogs, living in houses in an area of L. braziliensis transmission presented cutaneous and mucosal ulcers similar to the one presented by humans. In all the houses with canine CL there were active or past history of cases of human CL, and L. braziliensis isolated from dogs were genetically similar to the one from humans. These data are in favor of the participation of dogs in the transmission of L. braziliensis and may have a great importance in the control of the expansion of L. braziliensis infection. | 5,115 | 257 | lay_plos | en |
Summarize: By. Laura Collins. PUBLISHED:. 21:18 EST, 19 November 2012. |. UPDATED:. 21:32 EST, 19 November 2012. It is the scandal that has shaken the community of Kennebunk, Maine, to its core. One month since Alexis Wright was charged with running a brothel from her Zumba studio in the genteel New Hampshire town, local police continue to work through her client list and the revelations keep on coming. Once known primarily for its. pristine beaches and proximity to the Bush family's summer residence in. Kennebunkport, the scandal has rocked the New Hampshire town to its very. foundations in more ways than one. Scroll down for video. Energetic and fun loving: Alexis Wright, 29, in more sombre mood as she is arraigned on 106 charges last month. Showing a different side: sultry and fun loving Alexis in a photograph shot by promoter, Mark Strong. Because in a bizarre twist it has now emerged that as many as half of the 56 men charged to date work or worked in a business associated with home building, planning or construction. Notable among them is Donald Fortin,. 59, owner and founder of Fortin Construction Inc, a company that boasts. of being 'Number one Maine Home Builder for over 20 years,' and advertises itself online as 'A Christian Family Owned Company.' Indeed Mr Fortin is such a staunch. advocate of conservative family values. According to state records, he. donated $1000 to Protect Maine Marriage and organisation set up to. 'defend' marriage in the state from the perceived onslaught from 'gay. rights activists' and 'radical judges' seeking to'redefine marriage'. He also made a donation of $350 to Senator Garrett Mason a conservative opponent of the state's same-sex marriage ballot. Maine's 'Number One Home Builder,' and family values advocate, Donald Fortin, 59, has been charged by police. Invested in the community: Attorney Joseph Lewis, former Portland Planning Board member and Little League coach has also been implicated. Similarly vocal in his defence of family values is attorney Joe Lewis, identified by Kennebunk Police as one of the men charged with 'engaging a prostitute,' and a Portland based lawyer. Mr Lewis, 49, served on the Portland Planning Board from 2008 until summer 2012. In his professional biography he makes great play of his commitment to the community and to his family who are, he says, 'firmly settled in Portland'. He goes on: 'I can honestly say that of all the wonderful places I've had the chance to live in, Portland, ME is exactly where I want to be. 'I'm heavily invested in the community I served on the Portland Zoning Board of Appeal from 2000-2005. I also served on the Portland Planning Board from 2008 until summer, 2012.' He has coached and umpired for the local little League and volunteered in local schools. When approached by MailOnline Mr Lewis declined to comment. Speaking to Portland's Morning Sentinel, Ronald Weitzer, a sociologist at George Washington University and expert on prostitution said that the fact that a large number of Wright's alleged clients were in such related or similar businesses, 'does suggest that there was some referring going on.' Though truth be told it hardly takes an expert to draw such a conclusion. Alexis Wright is said to have kept meticulous notes, a 'Domination Fettish Sheet' and stands accused of having filmed clients in her Zumba studio and rented office. Former Mayor, James Soules, 58, a married father of four with 'lots of explaining to do' to his wife. Attorney Jens Bergen, 54, a 'happy guy who loves his wife and children' Financier Joe Cuetara,63, who lives in a $500,000 waterfront New Hampshire home. Gary Bahklow, 57, a realtor with a string of wealthy clients in search for real estate and timberland. Wright kept meticulous records suggesting a business enterprise that brought in more than $150,000 in 18months and including a 'Domination Fettish Sheet,' and client list reportedly running to 150 'johns.' This, along with other materials removed from her Zumba studio has formed the basis for much of the Kennebunk Police investigation. An honours student who raised money for good causes, none suspected Alexis Wright of living a double life. Speaking to Mail Online Lieutenant Antony Bean Burpee admitted that they were a long way from completeing their enquiries. He said: 'We've been investigating since September 2011 and we're nowehere near finished. 'We have to establish probably cause before we can charge these men and we're working through a long list.' The investigation was sparked by a tip off from a 'concerned resident' who had grown suspicious of the frequent male visitors to Wright's dance studio and, later, a rented office across the street. But nobody could possibly have imagined the torrent of allegation and counter-allegation that would follow. To most of those who knew her to that point Wright was simply 'a fun and energetic' girl, a former honours student from neighbouring Wells who took part in charity drives and class trips with her 7-year-old son. She hosted Zumba charity events for earthquake victims in Haiti, the Susan G Komen foundation for breast cancer and Toys for Tots. These are the sort of our-of-hours activities residents of the light-house studded coastline expect of their neighbours. But if the charges against are prove true she did all this while leading a double life that included acts of prostitution and filming clients. So far police have released three. batches of names of men charged with engaging a prostitute. As well as. the peculiar prevalence of contractors, painters, drywall and concrete specialists,. window and flooring professionals, realtors, appraisers and real estate. attorneys the list has included former Mayor James Soule, 58, realtor. Gary Bahklow, 57, attorney Jens. Bergen, 54, financier Joe Cuetara, 63 and local ice hockey coach, Donald. Hill who has since stepped down from his role at Kennebunk High. All. of the accused men sought to prevent their names being made a matter of. public record in an application that was last month denied in. Cumberland County Superior Court by Justice Thomas Warren. Picture Perfect: Kennebunk had long been known simply for its white beaches, light-house studded coastline, lobster and proximity to the Bush family's summer home. They are not the only men seeking to distance themselves from Alex Wright, the Zumba instrutcor who allegedly employed a number of online aliases to advertise her willingness to 'have fun(Sex) with both men and women'. The lawyer acting for Mark Strong, 57, Wright's former business partner and alleged co-conspirator, has applied to have Strong's case heard separately from the dance instructor. According to Strong's lawyer, Daniel Lilley, this is because Wright faces far more serious felony charges than Strong who has been charged with 59 misdemeanours - all of which he denies. She has been charged on 106 counts of prositution, invasion of privacy, conspiracy, tax offences and receving welfare benefits when ineligible for welfare. Mr Lilley said, 'It is guilt by association and we woudl just as soon go this month on our own, by ourselves.' Mr Lilley is also hoping that his client's trial can be heard before Christmas - an impossiblity for Wright as the police continue to issue summonses, and release names, pushing the trial well into next year | Summary: Hypocrisy of a Hallmark community as the men who built Kennebunk dominate list of those accused in the scandal rocking it. Main's 'Number One Home Builder,' a family values advocate who promotes his business as' Christian' and 'Family owned,' is charged. Little League Coach and Portland Planning Board member also on list of 'clients' charged. Alexis Wright's business partner and co-accused complains of 'guilt by association' | 1,801 | 101 | cnn_dailymail | en |
Write a title and summarize: SECTION 1. SHORT TITLE; AMENDMENT OF 1986 CODE. (a) Short Title.--This Act may be cited as the ``Internet Gambling Regulation and Tax Enforcement Act of 2009''. (b) Amendment of 1986 Code.--Except as otherwise expressly provided, whenever in this Act an amendment is expressed in terms of an amendment of a section or other provision, the reference shall be considered to be made to a section or other provision of the Internal Revenue Code of 1986. SEC. 2. TAX ON INTERNET GAMBLING; LICENSEE INFORMATION REPORTING. (a) In General.--Chapter 36 (relating to certain other excise taxes) is amended by adding at the end the following new subchapter: ``Subchapter E--Internet Gambling ``Sec. 4491. Imposition of Internet gambling license fee. ``Sec. 4492. Record requirements. ``SEC. 4491. IMPOSITION OF INTERNET GAMBLING LICENSE FEE. ``(a) Federal Fee.--Each licensee within the meaning of section 5382 of title 31, United States Code, shall be required to pay an Internet gambling license fee by the end of each calendar month in an amount equal to two percent of all funds deposited by customers during the preceding month into an account maintained by that licensee or any agent of that licensee that can be used for the purpose of placing a bet or wager as defined in section 5362(1) of title 31, United States Code. ``(b) Deposits.--Deposits made by or on behalf of a licensee of Internet gambling winnings or returns of funds by or on behalf of a licensee to the account of a customer shall not be treated as a deposit for purposes of this section. ``(c) Persons Liable for Fee.--The Internet gambling license fee shall be the direct and exclusive obligation of the Internet gambling operator and may not be deducted from the amounts available as deposits to the person placing a bet. Notwithstanding the foregoing, any person making a deposit for the purpose of placing a bet or wager with a person who is required but has failed to obtain a license pursuant to subchapter V of chapter 53 of title 31, United States Code, shall be liable for and pay the fee under this subchapter on all such deposits, but such liability shall not excuse any failure to pay the fee on the part of the person who is required but has failed to obtain such license. ``(d) Unauthorized Bets or Wagers.--There is hereby imposed a fee in an amount equal to 50 percent of all funds deposited into an account that can be used for placing a bet or wager within the meaning of Section 5362(1) of title 31, United States Code, with any person that is not authorized pursuant to section 5382 of that title. Such tax is due by the end of each calendar month with respect to deposits during the preceding month. ``(e) Disposition.--Amounts paid as Internet gambling license fees or on unauthorized bets or wagers under this section shall be deposited in the general fund of the Treasury and treated as revenue. ``(f) Administrative Provisions.--Except to the extent the Secretary shall by regulations prescribe, the fees imposed by this section shall be subject to the administrative provisions of this title applicable to excise taxes imposed by chapter 35. ``SEC. 4492. RECORD REQUIREMENTS. ``Each person liable for fees under this subchapter, except for a person making a deposit who is liable for fees pursuant to section 4491(e), shall keep a daily record showing deposits as defined in this subchapter, in addition to all other records required pursuant to section 6001(a).''. (b) Information Returns.--Subpart A of part III of subchapter A of chapter 61 (relating to information concerning persons subject to special provisions) is amended by adding at the end the following new section: ``SEC. 6050X. RETURNS RELATING TO INTERNET GAMBLING. ``(a) Requirement.--Every person who is a licensee (within the meaning of section 5382(3) of title 31, United States Code) or who otherwise is engaged in the business of accepting any bet or wager within the meaning of section 5362(1) of title 31, United States Code, during a taxable year shall furnish, at such time and in such manner as the Secretary shall by regulations prescribe, the information described in subsection (b), and such person shall maintain (in the location, in the manner, and to the extent prescribed in regulations) such records as may be appropriate to the information described in subsection (b). ``(b) Required Information.--For purposes of subsection (a), the information described is set forth below, which information may be modified as appropriate by the Secretary through regulation-- ``(1) the name, address, and TIN of the licensee or other person engaged in the business of accepting any bet or wager, ``(2) the name, address, and TIN of each person placing a bet or wager with the licensee or other person engaged in the business of accepting any bet or wager during the calendar year, ``(3) the gross winnings, gross wagers, and gross losses for the calendar year of each person placing a bet or wager with the licensee or other person engaged in the business of accepting any bet or wager during the year, ``(4) the net Internet gambling winnings for each such person for the calendar year, ``(5) the amount of tax withheld with respect to each such person for the calendar year, ``(6) beginning and end-of-year account balances for each such person for the calendar year, and ``(7) amounts deposited and withdrawn by each such person during the calendar year. ``(c) Statement To Be Furnished to Persons With Respect to Whom Information Is Required.--Every person required to make a return under subsection (a) shall furnish to each person whose name is required to be set forth in such return by reason of placing a bet or wager a written statement showing-- ``(1) the name, address, and phone number of the information contact of the person required to make such return, and ``(2) the information required to be shown on such return with respect to each person whose name is required to be set forth in such return. The written statement required under the preceding sentence shall be furnished to the person on or before January 31 of the year following the calendar year for which the return under subsection (a) was required to be made. ``(d) Definitions.-- ``(1) Net internet gambling winnings.--The term `net Internet gambling winnings' means gross winnings from wagers placed over the Internet with a person required to be licensed under section 5382 of chapter 53 of title 31, United States Code, less the amounts wagered. ``(2) Internet; wager.--The terms `Internet' and `wager' shall have the respective meanings given such terms by section 5362 of chapter 53 of title 31, United States Code.''. (c) Clerical Amendments.-- (1) The table of subchapters for chapter 36 is amended by adding at the end the following new item: ``subchapter e. internet gambling.''. (2) The table of sections for subpart B of part III of subchapter A of chapter 61 is amended by inserting after the item relating to section 6050W the following new item: ``Sec. 6050X. Returns relating to Internet gambling.''. (d) Effective Date.--The amendments made by this section shall apply to bets or wagers placed after the date of the enactment of this Act. SEC. 3. WITHHOLDING FROM CERTAIN GAMBLING WINNINGS. (a) Net Internet Gambling Winnings.--Paragraph (3) of section 3406(b) (relating to other reportable payments for purposes of backup withholding) is amended-- (1) by striking ``or'' in subparagraph (E); (2) by striking ``.'' and inserting ``, or'' at the end of subparagraph (F); and (3) by adding at the end thereof the following new subparagraph: ``(G) section 6050X(b)(4) (relating to net Internet gambling winnings).''. (b) Effective Date.--The amendment made by this section shall apply to bets or wagers placed after the date of the enactment of this Act. SEC. 4. WITHHOLDING OF TAX ON NONRESIDENT ALIENS. (a) Tax on Nonresident Alien Individuals.--Paragraph (1) of section 871(a) (relating to income not connected with United States business) is amended-- (1) by striking ``and'' at the end of subparagraph (C), (2) by inserting ``and'' at the end of subparagraph (D), and (3) by inserting after subparagraph (D) the following new subparagraph: ``(E) the gross amount of winnings from each wager placed over the Internet with a person required to be licensed under section 5382 of chapter 53 of title 31, United States Code (as such terms are defined in section 6050X(d)(2)),''. (b) Exemption for Certain Gambling Winnings.--Section 871(j) (relating to exemption for certain gambling winnings) is amended by inserting before the period at the end the following: ``or to any bets or wagers placed over the Internet (as such terms are defined in section 6050X(d)(2))''. (c) Withholding of Tax on Nonresident Alien Individuals.--The first sentence of subsection (b) of section 1441 (relating to withholding of tax on nonresident aliens) is amended by inserting after ``gains subject to tax under section 871(a)(1)(D),'' the following: ``the gross amount of winnings from wagers placed over the Internet described in section 871(a)(1)(E),''. (d) Source of Internet Gambling Winnings.--Subsection (a) of section 861 is amending by inserting at the end thereof the following new paragraph: ``(9) Internet gambling winnings.--Any Internet gambling winnings received from a licensee within the meaning of section 5382(3) of title 31, United States Code.''. (e) Effective Date.--The amendments made by this section shall apply to bets or wagers placed after the date of the enactment of this Act. SEC. 5. TERRITORIAL EXTENT. Paragraph (2) of section 4404 is amended to read as follows: ``(2) placed within the United States or any Commonwealth, territory, or possession thereof by a United States citizen or resident.''. | Title: To amend the Internal Revenue Code of 1986 to regulate and tax Internet gambling Summary: Internet Gambling Regulation and Tax Enforcement Act of 2009 - Amends the Internal Revenue Code to: (1) impose an Internet gambling license fee on Internet gambling operators and an additional tax on unauthorized bets or wagers; (2) require such operators to file informational returns identifying themselves and the individuals placing bets or wagers with them; (3) require withholding of tax on net Internet gambling winnings and on the winnings of nonresident aliens; and (4) extend the excise tax on wagers to include wagers placed within the United States or any commonwealth, territory, or possession by a U.S. citizen or resident. | 2,605 | 161 | billsum | en |
Summarize: Gary Dawn, who is also known as Pogo the Clown, who is accused of viciously attacking a police officer with a bottle of vodka. A professional clown who was accused of viciously attacking a police officer with a bottle of vodka has walked free from court after being found not guilty. Gary Dawn, who is also known as Pogo the Clown, was accused of maliciously wounding PC Mark Szeremetta leaving him hospitalised with a gaping wound to his skull. But today a jury at Nottingham Crown Court found the 54-year-old not guilty of the offence. The court had heard how the 54-year-old had been drinking on February 12 after the break-up of his long term relationship, which had left him feeling depressed. But when a friend called police over concerns for his welfare, Dawn lashed out when the officer arrived at the scene in Stoke Bardolph near Gedling in Nottinghamshire. The court heard how PC Szeremetta approached the accused before he launched the bottle of vodka he was drinking in the officer's face. The officer was then taken to hospital with a head injury and needing the wound glued, jurors were told. Prosecutor Mark Knowles told the court Dawn's actions were'reckless' and that he intended to do harm. He said: 'It's a straight forward issue. He pushed the officer away and the Crown will say that's indicative of the defendant's mood. 'He had no regard for others.' Giving evidence, PC Szeremetta, who was first on the scene on Stoke Lane said: 'I got out of the police vehicle and went to approach the male. 'He had his back to me at that stage.' PC Szeremetta said after shouting after Dawn he then approached the defendant when he was struck by the bottle. He added: 'I was very shocked, I was blinded, I could not really see at the time what I had been struck with. Dawn denied the charge at Nottingham Crown Court, pictured, and the trial continues. 'My priority was the preservation of the male.' Jurors were told the police officer caught up with Dawn - who has previously made attempts at taking his own life - and sat on him until other officers arrived. Dawn, who has been a professional clown for 20 years and done charity work with When You Wish Upon A Star, told the court: 'I was not thinking of the consequences. 'I was just down, looking down. 'There was someone behind me and I got frightened and scared.' Dawn denied the charge claiming it wasn’t intentional and he was just trying to push the officer away without realising the bottle was in his hand. He added he only realised the hand he struck the officer with had a bottle in it was when his 'hand felt lighter' | Summary: Gary Dawn, also known as Pogo the Clown, accused of attacking PC. Court heard how he had been drinking following a relationship break-up. PC Mark Szeremetta approached him after concerns for his welfare. Alleged he then launched a bottle of vodka in the police officer's face. PC left with a gaping wound to his skull and needing hospital treatment. But Dawn, who denied the charge was found not guilty by the jury. | 633 | 99 | cnn_dailymail | en |
Write a title and summarize: Comey dijo ante el Comité de Inteligencia del Senado que se sintió "confundido" por los cambiantes motivos sobre su despido. Durante las dos horas y media de declaración pública frente al Comité de Inteligencia del Senado, Comey ha acusado al gobierno de Trump de mentir en varias oportunidades. "Puedo decir con certeza que el presidente no es un mentiroso", dijo este jueves Sarah Sanders, vocera de la Casa Blanca, según la periodista de la BBC en Washington DC Tara McKelvey. El Comité de Inteligencia del Senado está investigando los supuestos vínculos de la campaña de Trump con funcionarios rusos en los meses previos y posteriores a las elecciones presidenciales de noviembre de 2016. Comey fue despedido el 9 de mayo por Trump, un anuncio que sacudió el corazón político de Estados Unidos. Mientras fue director del FBI, Comey creó memorandos de cada reunión que tuvo con Trump. El FBI también está investigando la intromisión de Rusia en las elecciones estadounidenses y los supuestos vínculos de la campaña de Trump con el gobierno de Moscú, indagaciones que Comey dirigía al momento de su remoción. "Me preocupaba que pudiera mentir" De forma conciliadora, este jueves Comey comenzó diciendo que entiende que el presidente tiene derecho a despedir a un director del FBI en cualquier momento por cualquier razón "o ninguna" antes de terminar su período de 10 años. Pero, agregó, que los motivos de su despido, los cuales fueron cambiando a lo largo de los días, lo hicieron sentir "confundido y cada vez más preocupado". Especialmente cuando, a través de los medios, se enteró que Trump lo removió del cargo por la "investigación de Rusia": "Eso no tenía sentido para mí", acotó. La audiencia pública ante el Comité de Inteligencia del Senado duró dos horas y media. Luego, Comey declaró a puertas cerradas. La versión original que brindó el gobierno para su despido fue que Comey no era capaz de liderar el FBI y que, como consecuencia, el buró había perdido la confianza en su director y que "era un desorden". "Esas fueron mentiras puras y llanas", afirmó Comey. En este sentido, el jurista republicano de 56 años también explicó por qué realizó memorandos tras cada conversación que mantuvo con Trump. "Honestamente me preocupaba que pudiera mentir acerca de la naturaleza de nuestra reunión, así que pensé que era realmente importante documentarla", dijo y aclaró que no realizó memorandos tras sus contadas conversaciones con los expresidentes Barack Obama y George W. Bush. Comey incluso confirmó que él le pidió a un "amigo cercano" (un profesor de la Escuela de Derecho de Columbia, dijo) que compartiera los memorandos sobre Trump con un periodista. Donald Trump y su equipo desmintieron que el presidente le haya dicho a Comey: "Yo necesito lealtad, yo espero lealtad". Su intención, agregó, era "guiar" hacia la designación de un investigador especial sobre la posible interferencia de Rusia en las elecciones presidenciales de EE.UU. de 2016. El 17 de mayo el exjefe del FBI Robert S. Mueller III fue nombrado para el puesto. Palabra contra palabra "No tengo dudas", dijo Comey cuando le preguntaron si hackers rusos intentaron interferir en las elecciones presidenciales con el conocimiento de "lo más alto" del gobierno de Moscú. Pero, ¿consiguieron alterar los votos en sí? "No", respondió. Durante la audiencia, que fue seguida de cerca por muchos estadounidenses, Comey confirmó que mientras estuvo a cargo del FBI, Trump jamás estuvo investigado personalmente por posibles vínculos con Rusia. La audiencia pública fue seguida de cerca por muchos estadounidenses, algo que llamó la atención de los analistas. El exdirector del FBI reconoció que mucho de lo que afirma, especialmente en los memorandos, enfrenta su palabra contra la de Trump. Por eso, dijo desea que "haya cintas" de lo que hablaron, algo que el propio presidente sugirió en mayo. "Espero que no haya grabaciones de nuestras conversaciones", escribió Trump en su cuenta de Twitter, una inusual advertencia del mandatario sobre la filtración de información a la prensa. ¿"Ordena" o "espera"? Este miércoles, el día previo a su declaración, Comey envió al comité su testimonio oficial describiendo todas las conversaciones privadas que mantuvo con el presidente estadounidense. Comey confirmó bajo juramento que, mientras fue director del FBI, el buró no investigó a Trump personalmente por posibles vínculos con Rusia. Allí explica, por ejemplo, que durante una cena privada en la Casa Blanca el pasado 27 de enero, Trump le dijo: "Yo necesito lealtad, yo espero lealtad". Esta conversación ha sido desmentida por la Casa Blanca. En dicha declaración previa, Comey también explicó los detalles de una reunión en la que el mandatario le pidió que el FBI "dejara pasar" la investigaciónque estaba realizando de los vínculos del exasesor de Seguridad Nacional, Michael Flynn, con Rusia. El abogado personal de Trump, Marc Kasowitz, dijo este mismo jueves que el presidente "jamás sugirió o instruyó" a Comey que dejara de investigar a nadie, algo que la Casa Blanca también ha reiterado en distintas oportunidades. El exasesor de Seguridad Nacional, Michael Flynn, estuvo solo 24 días en el cargo. En la audiencia, el senador republicano Marco Rubio le preguntó a Comey si efectivamente Trump le "ordenó" que abandonara la indagación, a lo que el ex director del FBI respondió que no. En este sentido, el hijo mayor del presidente y una de las cabezas de la Organización Trump, Donald Trump Jr., escribió en su cuenta oficial de Twitter: "Conozco a mi padre desde hace 39 años y puedo decir que cuando él 'ordena o dice' que hagas algo no hay ambigüedad, sabes exactamente a lo que se refiere". Flynn estuvo solo 24 días en el cargo ya que renunció por las revelaciones de que tuvo contactos con el embajador ruso, Sergei Kislyak, a quien le aseguró que EE.UU. levantaría las sanciones económicas sobre su país. Según el testimonio escrito de Comey, el 14 de febrero acudió a la Oficina Oval a un encuentro sobre lucha contra el terrorismo, en el que participaron varios altos funcionarios entre los cuales se encontraba el fiscal general, Jeff Sessions. El presidente de Rusia, Vladimir Putin, con una gorra de la campaña de Donald Trump, durante recientes protestas en Nueva York. Al concluir la reunión, Trump pidió a las demás personas que salieran de la habitación, incluyendo a Sessions. "Quiero hablar sobre Michael Flynn", dijo el mandatario cuando se quedaron a solas. A continuación comentó que Flynn no había hecho nada malo en sus conversaciones con los rusos, pero que había tenido que despedirlo porque no había informado adecuadamente al vicepresidente, Mike Pence. "Él (Flynn) es un buen hombre. Espero que puedas dejar pasar esto", afirmó Trump. Durante la audiencia pública, Comey dijo que, como investigador, si tuviera que determinar quién dice la verdad, tomaría en cuenta el hecho de que Trump solicitó tener la conversación a solas. "Ese es un dato muy significativo para mí. ¿Por qué necesitaba echar a todo el mundo de la habitación?", declaró. | Title: El gobierno de Trump dijo "mentiras puras y llanas": James Comey, exdirector del FBI, declara frente al Senado en investigación sobre vínculos entre la campaña del presidente de EE.UU. y Rusia Summary: "El gobierno (de Donald Trump) decidió difamarme a mí y, lo que es más importante, al FBI", dijo James Comey, exdirector del Buró Federal de Investigaciones, este jueves de mañana en su audiencia frente al Senado de Estados Unidos. | 1,576 | 103 | xlsum_es | es |
Summarize: Intel is officially announcing its next-generation processor tomorrow--and Hollywood is playing a big role. Sandy Bridge--or what Intel now calls the 2nd Generation Intel Core processor family--is a big step for the chipmaker because, for the first time in a mainstream product, the graphics chip is grafted directly onto the main processor, boosting performance. This design essentially provides the graphics function for free, allowing PC makers to bring out laptops that don't have to always rely on separate graphics processors from Advanced Micro Devices or Nvidia. (For more on the technical details of the new chip, see this companion report.) "It's all about the visual experience and smarter performance," Tom Kilroy, senior vice president and general manager of Intel's Sales and Marketing Group, said in an interview last week. "It's well documented how online video is going on across the globe. There's over 100 million people doing user-generated content creation. So, what we tried to do with the architecture is really improve that experience. We're calling it the User Visual Experience." One of Sandy Bridge's marquee features is high-speed, on-the-fly conversion between data formats. The chip also includes the next version of Intel's Turbo Boost--version 2.0, a key technology that speeds up and slows down the processor to optimize performance and power, respectively. Sandy Bridge highlights: 20 new Core i3, i5, i7 processors: First chips available (January) in laptops will be quad-core. First chips available (January) in laptops will be quad-core. More than 500 new PC systems: coming from PC makers worldwide. coming from PC makers worldwide. Enhanced graphics silicon: built directly onto the main processor. built directly onto the main processor. Quick Sync Video: accelerates encoding/decoding of media formats. accelerates encoding/decoding of media formats. Turbo Boost 2.0: power-efficient overclocking of processor. power-efficient overclocking of processor. Intel Wireless Display 2.0: WiDi 2.0 beams content to big-screen HDTVs at 1080pHD resolution. WiDi 2.0 beams content to big-screen HDTVs at 1080pHD resolution. Intel Insider: direct access to movies, not available before, on PCs in high definition. Access to Hollywood content is also baked into the chip--a technology called Intel Insider. "This will unlock premium high-definition content, like movies, to your PC," Kilroy said. "We've gone out and engaged with the studios. So, you'll see Warner Bros. and Fox at launch [of Sandy Bridge] and several other studios to come. They're eagerly embracing this platform as a distribution means for premium high-end content--as Internet content [offered] directly to the end user." Kilroy continued. "What Intel Insider does is deliver HD digital distribution rights to the PC. This could be enabled through multiple content storefronts through OEMs (PC makers), retailers like Best Buy. Essentially, the PC now becomes an on-ramp for HD 1080p movies," he said. And Intel has added security features to protect the content. "And we've built in security capability into this platform that will enable end-to-end hardware protection for the content. So, it will protect the premium content rights of the studios," according to Kilroy. Among the hundreds of new systems expected to flood the market over the next several months, Hewlett-Packard and Dell will come out with high-end quad-core laptops. Other dual-core systems will follow in February, according to Intel. "The quad core based systems will be available on January 9 and additional dual-core versions in February," Intel said in a statement. Many new Sandy Bridge systems will be demonstrated at the Consumer Electronics Show, which begins on January 6 in Las Vegas. Intel Corp.'s latest chip line is arriving this week with a surprise link to Hollywood: security technology that has persuaded some companies to let personal-computer users view movies and television shows in a top-quality video format for the first time. The Silicon Valley giant is announcing the new chips at the Consumer Electronics Show in Las Vegas this week, along with deals with companies that distribute video content over the Internet. Though they have long let PC users view or download videos—including movies labeled as high-definition—piracy concerns have deterred studios from offering content in a particularly high-resolution format called 1080p... | Summary: Intel has added a surprise new feature to its next generation of Core microprocessors-an anti-piracy feature that it hopes will convince studios to stream true 1080p videos. Until now, studios have offered "HD" streaming content, but they've held back on the true 1080p resolution out of fear of pirates, the Wall Street Journal explains. Intel's new chips-codenamed "Sandy Bridge"-will have an "Intel Insider" feature that is intended to ensure the processor won't copy such content. Intel will debut the chips at CES this week, and announce video distribution deals with several companies, including Warner Bros. and Fox. "The new Intel technology is a fundamental change for us," says one Warner Bros. executive. "It creates a fundamentally more secure platform in the PC environment." On the technical side, the chips also take a big step by grafting a graphics chip directly onto the main processor, which should improve visual performance and eliminate the need for a separate graphics card, CNet reports. | 994 | 223 | multi_news | en |
Write a title and summarize: Trachoma is a blinding disease, initiated in early childhood by repeated conjunctival infection with the obligate intracellular bacterium Chlamydia trachomatis. The population prevalence of the clinical signs of active trachoma; ‘‘follicular conjunctivitis” (TF) and/or ‘‘intense papillary inflammation” (TI), guide programmatic decisions regarding the initiation and cessation of mass drug administration (MDA). However, the persistence of TF following resolution of infection at both the individual and population level raises concerns over the suitability of this clinical sign as a marker for C. trachomatis infection. We systematically reviewed the literature for population-based studies and those including randomly selected individuals, which reported the prevalence of the clinical signs of active trachoma and ocular C. trachomatis infection by nucleic acid amplification test. We performed a meta-analysis to assess the relationship between active trachoma and C. trachomatis infection before and after MDA. TF and C. trachomatis infection were strongly correlated prior to MDA (r = 0. 92,95%CI 0. 83 to 0. 96, p<0. 0001); the relationship was similar when the analysis was limited to children. A moderate correlation was found between TI and prevalence of infection. Following MDA, the relationship between TF and infection prevalence was weaker (r = 0. 60,95%CI 0. 25 to 0. 81, p = 0. 003) and there was no correlation between TI and C. trachomatis infection. Prior to MDA, TF is a good indicator of the community prevalence of C. trachomatis infection. Following MDA, the prevalence of TF tends to overestimate the underlying infection prevalence. In order to prevent unnecessary additional rounds of MDA and to accurately ascertain when elimination goals have been reached, a cost-effective test for C. trachomatis that can be administered in low-resource settings remains desirable. Sight loss from trachoma is the end result of a scarring disease process that is initiated in early childhood by the obligate intracellular bacterium Chlamydia trachomatis [1]. Repeated infection of the conjunctiva by C. trachomatis causes a recurrent chronic follicular conjunctivitis (TF) of the upper eyelid mucosal surface (Fig 1) [2]. This can sometimes be particularly severe with intense papillary inflammation (TI). Together, TF and / or TI are collectively referred to as “Active Trachoma”. Conjunctival scarring gradually develops, usually becoming visible from early adulthood. Eventually the scarring causes the eyelashes to turn in and scratch the surface of the eye. If left uncorrected, trichiasis traumatises the cornea surface, resulting in opacification and sight loss. There is a variable relationship between the clinical signs of Active Trachoma and the presence of C. trachomatis infection. The natural history of an infection episode in children is probably characterised by a brief “pre-clinical” phase, in which there is detectable infection but the clinical signs of the inflammatory response are yet to develop (Fig 1). Human volunteer experiments in which the conjunctiva was inoculated with C. trachomatis indicated that the signs of disease typically take about 10 days to develop in previously uninfected individuals [3]. This is followed by a variable period of time in which both infection and disease can be detected at the same time; this may last for several days to many weeks. The immune response brings the infection under control, completely clearing it or reducing it to undetectable levels. However, inflammatory clinical signs persist, typically lasting several weeks after the resolution of the infection. In children aged 4–15 years, data from longitudinal cohort studies estimate the median duration of infection range between 23 days and 8 weeks, and the median duration of disease between 54 days and 18 weeks [4–6]. The duration of disease and infection declines with increasing age. Therefore, at an individual level there is frequently a large mismatch between when infection can be detected and the clinical signs of disease are found [7]. The most recent estimates from the World Health Organization (WHO) Alliance for the Global Elimination of Trachoma by 2020 (GET2020) estimates indicate that about 200 million people live in trachoma endemic areas in 42 countries, 2. 2 million have visual impairment or blindness, and about 3. 2 million have trichiasis [8]. To meet this large public health challenge, the GET2020 Alliance recommends the implementation of the SAFE Strategy which tackles the disease at different stages: Surgery to correct trichiasis, Antibiotics to treat chlamydial infection and Facial cleanliness and Environmental improvements to suppress transmission of the infection [1]. The antibiotic azithromycin is being used in community-wide mass drug administration (MDA); it is given as a single oral dose on an annual basis in endemic districts. Decisions around when to initiate and stop MDA are guided by the prevalence of TF in children in endemic communities. According to the current guidelines, azithromycin MDA is indicated for entire districts where the prevalence of TF in 1–9 year olds is ≥10%. Moreover, the determination of whether a program has controlled the active stage of trachoma as a public health problem also rests on the district level prevalence of TF [9]. Therefore, in view of the significance attached to TF for making programmatic decisions, it is important to understand the relationship between active disease and chlamydial infection in endemic communities. It is probable that this relationship changes after the introduction of MDA and will vary with different levels of endemicity. Several studies have specifically investigated what clinical signs can tell us about infection [7,10–19]. However, in addition there are many other population-based studies, which report on both disease and infection, that can contribute information. Here we systematically review the published literature for reports that can inform our understanding of the relationship between clinical signs of active disease and C. trachomatis infection, both before and after the introduction of MDA. In this review we included population-based studies and studies involving a random selection of participants that report the relationship between signs of Active Trachoma (TF, TI, TF/TI) and the detection of ocular C. trachomatis by nucleic acid amplification tests (NAAT), including polymerase chain reaction (PCR), polymerase chain reaction/enzyme immunoassay (PCR-EIA), ligase chain reaction (LCR) and transcription-mediated amplification (TMA). We excluded studies which did not test individuals without Active Trachoma for infection. References were identified through searching PubMed for articles using the terms (i) “trachoma” AND “Chlamydia trachomatis”, (ii) “Trachoma” AND “PCR”, (iii) “Trachoma” AND “LCR”, (iv) “Trachoma” AND “azithromycin”. The search was limited to 1991 onwards, the year of the first report of the use of PCR to test for ocular C. trachomatis infection in a trachoma endemic population [20]. The search was last updated on 5th May 2016. The titles and abstracts of all articles resulting from these searches were screened by two authors (AMR, MJB) for potentially eligible publications. These two authors then independently assessed the potentially eligible articles for inclusion and extracted the data. The bibliographies of publications meeting the inclusion criteria were also reviewed for any additional publications not already identified. Studies were excluded where the participant sample was not population-based. Several studies were identified with multiple related published reports arising from the same data. These are considered as a single study, but for completeness we include all the relevant references. Core information was extracted using a standardised form. The core information included country, year of publication, study design, study population size, age group, TF prevalence, TI prevalence, TF/TI prevalence (if TF alone was not reported), C. trachomatis infection prevalence, diagnostic test used, grading system, use of antibiotic for infection control. The clinical grading system was recorded: the 1981 Detailed WHO FPC System or the 1987 Simplified WHO Trachoma Grading System [2,21]. For the purpose of this review the prevalence levels of TF in the populations studied were categorised as follows: Hypoendemic <10%, Mesoendemic 10–20% and Hyperendemic >20% [12]. Due to the heterogeneity in the study designs and the reporting of data, only a limited meta-analysis was performed. Studies report the results for different age groups. Where available we separately present both the “All Age” results and those for children (≤15 years). In the meta-analysis we use the results from children only where these are available; where these are not available, the all age results are used. Summary graphs are presented of the relationship between: (i) the community prevalence of disease signs and C. trachomatis infection, (ii) the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) of TF for infection, by the community prevalence of disease signs. The degree of correlation between these was tested using Pearson’s correlation coefficient, weighted by study size. The relationship between signs and infection was analysed separately for pre-treatment and post-treatment data, then a test for interaction was performed on the two datasets combined to test whether the association between signs and infection was similar in the pre and post-treatment data. Forest plots were generated to illustrate the strength and heterogeneity of the relationship between disease signs and detection of infection; study heterogeneity was estimated using the I2 statistic. Hierarchical summary receiver operating characteristics (HSROC) curves were plotted to illustrate the relationship between the sensitivity and specificity of signs of TF for the presence of C. trachomatis infection and a pooled estimate of sensitivity and specificity was made in both the pre and the post MDA studies. All analyses were performed using Stata 13. A Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) checklist and flow diagram are included in the supporting information (S1 Checklist and S1 Fig). Of the 36 pre-treatment study populations, 24 were hyperendemic, 8 mesoendemic and 4 hypoendemic (Table 1). The studies were conducted in populations from Tanzania (12), The Gambia (7), Ethiopia (6), Nepal (4), Niger (3), Cameroon (1), Guinea Bissau (1), Egypt (1) and Australia (1). The Simplified WHO Trachoma Grading System was used in 28 studies and 8 used the Detailed WHO FPC System. The majority of studies (25) used the commercially produced Amplicor CT/NG (Roche) PCR assay, five used ligase chain reaction (LCR, Abbott Laboratories), and six used in-house PCR assays. Four of the 36 studies listed in Table 1 did not report the community prevalence of C. trachomatis infection, however they report infection on a selected subset of individuals [11,22–24]. These four studies were therefore retained in Table 1 as they contribute useful information, however they were excluded from the meta-analysis. Of the remaining 32 studies included in the meta-analysis, 29 studies reported data for childhood age groups (variable age ranges), seven of which also reported all age data, and three studies reported only all age data. Prior to the introduction of MDA there was evidence of a strong positive correlation between the community-level prevalence of Active Trachoma and the community-level prevalence of detected C. trachomatis, using data from all 32 studies (r = 0. 92,95%CI 0. 83 to 0. 96, p<0. 0001), Fig 2A. The correlation was similar when the analysis was limited to children only data (29 studies; r = 0. 91,95%CI 0. 82 to 0. 96, p<0. 0001). The correlation was much weaker for all age data (10 studies; r = 0. 51,95%CI -0. 18 to 0. 86 p = 0. 13). Overall, the community prevalence of TF is typically greater than the underlying community prevalence of detected C. trachomatis, with TF having a higher prevalence than C. trachomatis in 25 out of the 32 studies. The mean difference in prevalence was 10. 4% (95% CI 5. 9%-15. 0%, p = 0. 001). There were 19 studies that reported the prevalence of TI separately from TF. There was evidence of a moderate positive correlation between the community-level prevalence of TI and the community-level prevalence of detected C. trachomatis (r = 0. 75,95%CI 0. 45 to 0. 90 p = 0. 0002), Fig 2B. Although there are only a limited number of studies, in all communities where the TI prevalence was >20% the prevalence of C. trachomatis was at least 30%. In contrast, where the TI prevalence was below 20% there was a considerable degree of variation in the underlying community prevalence of infection. There was sufficient data at the individual level presented to estimate the sensitivity, specificity, PPV and NPV for 21 studies. The sensitivity of TF for identifying the presence of C. trachomatis infection varied substantially with the prevalence of TF in the community, Fig 3A. There was evidence of a strong positive correlation (r = 0. 82,95%CI 0. 59 to 0. 92, p<0. 0001). In contrast, there was a strong negative correlation between the specificity of TF for infection and the community prevalence of TF (r = -0. 92,95%CI -0. 97 to -0. 80, p<0. 0001), Fig 3B. The Forest plot of the pre-treatment relationship between disease and detection of infection at the individual level showed an overall strong association (OR 6. 05,95% CI 5. 49 to 6. 67, p<0. 0001), although there was marked heterogeneity (I2 = 87. 1%, p<0. 001), Fig 4A. A plot of sensitivity against specificity of the pre-treatment studies using TF as a test for C. trachomatis infection at the individual level showed that the overall estimated sensitivity was moderate (57. 1% (45. 4–68. 1%) and the overall specificity was good (81. 1% (73. 4–87. 0%), Fig 5A. The relationship between the community-level prevalence of Active Trachoma and the proportion of people with TF who were infected with C. trachomatis (Positive Predictive Value, PPV) showed a positive correlation (r = 0. 80,95%CI 0. 56 to 0. 91, p<0. 0001), Fig 3C. The pattern of this distribution suggests that when the community-level prevalence of TF is below 20% the PPV of TF for the presence of C. trachomatis infection drops substantially. Above a community-level prevalence of TF of 20% the PPV is typically 40–70%, across a wide range of TF prevalence. The relationship between the community-level prevalence of Active Trachoma and the proportion of people without TF who were not infected with C. trachomatis (Negative Predictive Value, NPV) showed a strong negative correlation (r = -0. 81,95%CI -0. 92 to -0. 57, p<0. 0001), Fig 3D. When the community-level TF prevalence was greater than 20%, the NPV was more variable. Of the 21 studies from populations following the introduction of MDA, prior to the first treatment, 15 were hyperendemic, 3 mesoendemic and 3 hypoendemic (Table 2). The studies were conducted in populations from Tanzania (10), Ethiopia (5), The Gambia (3), Nepal (2), and Egypt (1). The Simplified WHO Trachoma Grading System was used in 17 studies and four used the Detailed WHO FPC System. The majority of studies (15) used the Amplicor CT/NG (Roche) PCR assay, four used LCR (Abbott), one used an in-house PCR and one used the Aptima Combo2 (Hologic) TMA assay. There was one study included in Table 2 that did not report the community prevalence of C. trachomatis infection, and was therefore not included in the meta-analysis [23]. One of the Gambian studies involved surveys in four separate districts; we have included these as four separate sets of data in the analysis [25]. This resulted in 23 discrete studies included in the meta-analysis: for 20 studies data was available on children only (variable age ranges), two of which also reported all age data, and three other studies reported only all age data. Following the introduction of MDA there was evidence of a moderate positive correlation between the community-level prevalence of Active Trachoma and the community-level prevalence of detected C. trachomatis, using data from all 23 studies (r = 0. 60,95%CI 0. 25 to 0. 81, p = 0. 003), Fig 2C. The relationship was similar when the analysis was limited to the 20 studies of children only (r = 0. 60,95%CI 0. 21 to 0. 82, p = 0. 005). However, for the five studies reporting results for all ages there was no significant correlation (r = 0. 72,95%CI -0. 18 to 0. 86 p = 0. 18). The overall impression is that the relationship between disease and infection is more uncertain post-MDA, such that the community-level prevalence of TF can substantially overestimate the underlying community-level prevalence of C. trachomatis; the community prevalence of TF can remain high (>20%) even when the prevalence of infection has declined (<10%), Fig 2C and 2E. We found evidence that the relationship between the prevalence of TF and the prevalence of C. trachomatis infection differs between pre and post-MDA. Assuming a linear relationship between C. trachomatis and TF, the prevalence of C. trachomatis was associated with an expected 6. 5% increase for every 10% increase in TF in pre-MDA studies, compared with an increase of 2. 8% in post-MDA (test for interaction p = 0. 004). This demonstrates that as the prevalence of TF increases, the expected increase in C. trachomatis is higher in pre-MDA communities than in post-MDA communities. There were only eight studies that reported the prevalence of TI separately from TF after the introduction of MDA. There was no evidence of a correlation between the community-level prevalence of TI and the community-level prevalence of detected C. trachomatis (r = 0. 50,95%CI -0. 31 to 0. 89 p = 0. 20), Fig 2D. However, it is noteworthy that the prevalence of TI was low (<10%) in all but two communities. There was sufficient data presented to estimate the sensitivity, specificity, PPV and NPV for only 10 studies at the individual level after introduction of MDA. Therefore, only limited inference can be drawn. The sensitivity of TF for identifying the presence of C. trachomatis infection after the introduction of MDA varied widely, across a range of community-level TF prevalence levels, Fig 6A. In contrast, there was evidence of a strong negative correlation between the specificity of TF for infection and the community prevalence of TF (r = -0. 99,95%CI -0. 99 to -0. 96, p<0. 0001), Fig 6B. The Forest plot of the post-treatment relationship between disease and detection of infection at the individual level showed an overall strong association (OR 8. 38,95% CI 7. 09 to 9. 90, p<0. 0001), although there was marked heterogeneity (I2 = 75%, p<0. 001), Fig 4B. Although this overall OR is slightly higher post-MDA compared to pre-MDA, it should be noted that this result is calculated from individual-level data, whereas the correlations between TF and C. trachomatis infection prevalence (Fig 2) are calculated from population-level data. Compared to the pre-MDA situation (Fig 5A), after the introduction of MDA the individual level sensitivity was weaker (39. 2% (30. 9%, 48. 2%) while specificity was stronger (96. 1% (89. 6%, 98. 6%), Fig 5B. The community-level prevalence of Active Trachoma and the proportion of people with TF who were infected with C. trachomatis (PPV) did not appear to be correlated (r = 0. 16,95%CI -0. 52 to 0. 72, p = 0. 65), Fig 6C. Finally, the proportion of people without TF who were not infected with C. trachomatis (NPV) was high (>90%), across the limited range of community-level prevalence of Active Trachoma in these studies, Fig 6D. It has long been observed that the relationship between the signs of Active Trachoma and the detection of C. trachomatis infection at the individual level is not highly concordant [12]. Surveys, including those in this review, consistently find that within endemic populations there are many individuals with signs of disease who do not have detectable infection and conversely there are people who do not meet the diagnostic criteria for Active Trachoma (TF or TI) who do have detectable infection. Therefore, at the individual level, signs of infection cannot be depended upon to determine which members of an endemic community have ocular C. trachomatis infection. In a trachoma endemic population, the main reason for this mismatch between active disease and infection is probably the different time courses of the typical infection and disease episodes, outlined in the introduction and illustrated in Fig 1. In addition, it is also possible that other factors contribute to this mismatch. Some individuals who have detectable infection but do not meet the diagnostic criteria for TF may have a mild trachomatous follicular conjunctivitis. Others may have previously acquired immunity and are able clear the infection without developing any detectable inflammatory signs. Alternatively, a positive NAAT test for C. trachomatis in a clinically normal individual could arise through cross-contamination during sample collection or processing. Clinical signs similar to those of Active Trachoma can also arise for other reasons, such as viral or bacterial infections, vernal conjunctivitis and hypersensitivity reactions [26]. It is clear that clinical signs are not a reliable indicator for C. trachomatis at the individual level. There is no point-of-care diagnostic test available for programmes to use to determine which individual members of a community are infected, and who would therefore benefit from targeted antibiotic treatment. Moreover, a strategy of testing everybody is not considered a practical option. Therefore, the WHO guidance and the standard practice is to conduct community-wide antibiotic distribution of the entire population of endemic districts. Decisions around the initiation and cessation of trachoma control measures are based on the prevalence of TF in children aged 1–9 years, determined through district level surveys, such as those conducted by the Global Trachoma Mapping Project. District-wide antibiotic treatment programmes and F&E measures to suppress transmission are initiated where the initial prevalence of TF is ≥10%. Below 10% TF the advice is to conduct sub-district level surveys. If a sub-district has ≥10% TF MDA and F&E are implemented. For sub-districts between 5% and 9. 9% TF the guidance is to consider targeted MDA and F&E measures. For sub-districts <5% no MDA is needed and implementation of F&E can be considered. The dependence on the clinical signs of disease to guide programmatic decisions raises the important question of how accurate these clinical signs are as a proxy measure for C. trachomatis infection at the population level. This question is particularly relevant after the introduction of MDA, when the association between clinical signs and infection prevalence at the population level is weaker, and as we try to determine when elimination targets have been reached. In this systematic review, we found that prior to the introduction of antibiotic treatment the relationship between the community-level prevalence of TF in children correlated well with that of infection. The prevalence of TF was usually slightly greater than that of infection. Therefore, over a wide prevalence range, the community-level prevalence of TF in children is a reliable indicator that broadly reflects the underlying population burden of infection. The association between disease and infection for all ages was much weaker than that for children only; this supports the rationale for measuring TF in 1–9 year olds as the key indicator group for determining the need of antibiotic. The prevalence of TI was less well correlated than TF with infection before MDA, with the disease prevalence generally underestimating the level of infection. At the individual level the utility of TF as a marker for infection is highly sensitive to the underlying prevalence of TF. Both the sensitivity and PPV rise substantially with increasing TF prevalence, and the specificity and NPV both drop. This sensitivity increase is therefore offset by a corresponding decrease in the specificity of this test in high TF prevalence communities. The usefulness of TF as an indicator of an individual’s infection status is dependent on both the sensitivity and the specificity of the test; too low a sensitivity leads to C. trachomatis infected individuals not receiving treatment, whereas too low a specificity leads to wasted resources treating uninfected individuals. The PPV possibly gives the clearest indication of where TF is useful as an indicator of an individual’s infection status; when the prevalence of TF is low (~5–10%), TF will only indicate an estimated 10% probability that the individual has C. trachomatis infection. This probability increases steadily with prevalence and once the community prevalence reaches 30%, the estimated PPV is in the order of 50–70%. Thus, where the population TF prevalence is above 30%, a positive TF diagnosis gives a 50–70% probability that an individual will be C. trachomatis infected. After the introduction of MDA the relationship between the community prevalence of TF and chlamydial infection is less certain. Although the relationship between Active Trachoma and C. trachomatis infection appears to remain strong at the individual level, the population-level data suggests that post-MDA, Active Trachoma has a greater tendency to overestimate the underlying population prevalence of C. trachomatis infection. Below the 10% TF level the prevalence of infection was consistently low, and therefore below this level, TF prevalence appears to be a good marker for infection having being brought under control. However, in the studies where the prevalence of TF was above 10% after the introduction of MDA, the underlying prevalence of C. trachomatis infection was much less predictable. In some settings TF prevalence persists at high levels despite relatively little infection being detected. There might be several reasons for this observation. For example, if the loads of infection are substantially lower following the introduction of MDA these may not be so readily detected by diagnostic tests. Other bacterial species might also provoke a follicular conjunctivitis more readily in individuals previously infected with C. trachomatis [26]. However, whatever the explanation, it is likely that in some settings, the prevalence of TF will underestimate the impact MDA has had on the prevalence of C. trachomatis. This might lead to the on-going use of MDA after the infection has been adequately controlled. There is much less published data on the relationship between TI and infection following the introduction of MDA. In general, the prevalence of TI appears to reduce more readily than TF. A number of studies have investigated the relationship between the load of infection and disease signs. These suggest that TI is particularly associated with high loads of infection [27]. Therefore, the decline in TI may reflect a shift to less intense infections. However, there were a few studies in which the prevalence of TI was low, but the prevalence of infection remained substantial. We identified a reasonable number of studies reporting the community-level relationship between disease and infection prior to treatment, over a wide TF prevalence range. There were, however, fewer studies documenting the situation following the introduction of MDA, potentially limiting the conclusions that can be drawn after MDA. However, there was generally less detail in these reports about the individual-level relationship between clinical signs and infection. The studies came mostly from several East and West African countries, providing reasonable geographical coverage of the regions with the greatest trachoma burden. Standard WHO definitions of disease were generally used and the large majority of studies used the same commercially produced PCR assay, providing consistency across studies. However, there was some methodological heterogeneity. The age groups reported varied; where possible we use the data for children only in the meta-analysis to try to provide greater consistency between studies. The sample sizes varied considerably (from 56 to 7817); we weighted our analyses to adjust for size. The sampling methodology also varied considerably. The studies included were generally population-based samples or surveys of the entire resident population of a defined area. Overall, the use of TF prevalence to guide the decision to initiate MDA in previously untreated districts appears to be reliable. In contrast, the situation following treatment is more uncertain, calling into question the reliability of clinical signs for monitoring progress towards the achievement of the elimination targets [28]. There are reports from hyperendemic regions that have received many rounds of high-coverage MDA that suggest that the prevalence of TF can be recalcitrant, even when C. trachomatis infection appears to have been brought under control. Therefore, contextually appropriate, cost-effective tests for C. trachomatis infection that can be administered in low-resource settings, and used to estimate the infection prevalence in a population-based sample, would be very helpful in guiding decisions around the cessation of MDA. Such tests are anticipated to reduce the number of annual rounds of MDA required and lead to the confirmation of trachoma control at an earlier stage. | Title: The Relationship between Active Trachoma and Ocular Chlamydia trachomatis Infection before and after Mass Antibiotic Treatment Summary: Trachoma is the leading infectious cause of blindness worldwide, caused by the bacterium Chlamydia trachomatis. Repeated infection of the conjunctiva during childhood can initiate chronic conjunctival inflammation. This can lead to conjunctival scarring, in turning of the eyelashes, abrasion of the eyelashes on the cornea and eventually blindness later in adulthood. The World Health Organization recommends mass drug administration (MDA) for infection control when the prevalence of the clinical sign of Active Trachoma (TF) is ≥10% in 1-9 year olds. This systematic review of the literature examined the relationship between TF and C. trachomatis infection before and after MDA in order to evaluate the usefulness of TF for guiding trachoma control programmes. The population prevalence of TF and C. trachomatis infection were strongly correlated prior to MDA, however the relationship was weaker after MDA with a greater tendency for TF to overestimate the underlying infection prevalence. A cost effective test for C. trachomatis suitable for use in low resource settings could prevent unnecessary additional rounds of MDA in the population and could identify when trachoma elimination goals have been reached at an earlier time point. | 7,078 | 301 | lay_plos | en |
Summarize: WASHINGTON—With a bipartisan budget deal now on track to pass, the debt limit is looming as a focal point for the next high-stakes fiscal battle between Democrats and Republicans, as well as between tea-party and business-friendly factions of the GOP, as candidates position for the midterm elections. Conservative lawmakers and advocacy groups are pushing GOP leaders to make big demands of President Barack Obama early next year in... Washington (CNN) -- A federal budget compromise that already passed the House cleared a key procedural hurdle on Tuesday in the Senate, increasing the likelihood it will win final Congressional approval this week. President Barack Obama has signaled his support for the plan worked out by the budget committee leaders in each chamber that would guide government spending into 2015 to defuse the chances of another shutdown such as the one that took place in October. Tuesday's vote overcame a Republican filibuster attempt that required 60 votes in the 100-member chamber to proceed on the budget measure. The count was 67-33, with a dozen Republicans joining the 55 Democrats and independents in support of the plan. 5 ways Senate could mess it up Final approval in the Senate requires a simple majority of 51 votes. The budget plan easily passed the House last week on a 332-94 vote. GOP concerns The politics of the issue were clear in Tuesday's vote breakdown. Only one Republican Senator facing a primary challenge in their re-election campaigns voted with Democrats to overcome the GOP filibuster -- Lamar Alexander of Tennessee. Alexander has indicated he will oppose the budget plan in the final vote later this week. A new ABC News/Washington Post poll released Tuesday showed 50% of respondents approving of the budget plan while 35% opposed it. According to the survey, a majority of Democrats and independents backed the proposal, while only 39% of Republicans liked it. Some Senate Republicans said before Tuesday's vote that the most important thing for now was to lower the budget deficit, even if only by a small percentage, and prevent further government shutdowns like the 16-day stoppage in October that proved politically damaging to the GOP. Sen. Ron Johnson, who was lobbied to support the bill negotiated by fellow Wisconsin Republican Rep. Paul Ryan, said he wanted "to make sure we avoid any additional government shutdowns." "The federal government does enough harm to our economy," Johnson said. "We don't need to add additional harm by this crisis management. In the end this is not the kind of deal I would want to see. I'm sure it's not the kind of deal Paul Ryan would want to produce." Conservative GOP Sen. Orrin Hatch of Utah said in a statement that "sometimes the answer has to be yes." "Ultimately, his agreement upholds the principles conservatives stand for and, with Democrats controlling the White House and the Senate, it is the best we can hope for," he said of the plan Ryan negotiated with Democratic Sen. Patty Murray of Washington. CNN vote count: Budget deal nearing Senate approval, but not there yet Final congressional approval of the elusive budget agreement would mark a rare win for bipartisanship and a step up for a Congress infected with political dysfunction and held in low public esteem with midterm elections less than a year off. Democrats wary, too While Democrats supported the bill, many had concerns. More liberal senators -- like Tom Harkin of Iowa -- complained that an unemployment benefit extension was not included in the deal. "There's over a million people now who cannot find a job, out of work, and right at this time of year their unemployment insurance is being cut off," Harkin told Radio Iowa last week. "It's really unconscionable." Poll: Congress neck-and-neck with car salespeople, lobbyists for least honest Budget deal The budget agreement, which was months in the making, eases spending caps for the next two fiscal years while softening the impact of across-the-board spending cuts, known as the sequester, on defense and non-defense programs. Overall, it calls for more than $20 billion in deficit reduction. Current federal spending expires in mid-January, raising the possibility of another shutdown at that time if there's not a new agreement in place to keep federal coffers filled. The strong vote in the House on the budget plan on Thursday brought a collective sigh of relief among supporters, who initially thought it would sail through the Senate, where bipartisanship has been more the norm than in the sharply divided House. Don't expect Boehner to totally change his tune But after reading details of the agreement, many Senate Republicans -- including several in leadership positions -- came out against the bill. "I'd really like to stay within the (spending) caps," complained GOP Sen. John Boozman of Arkansas. "This busts the caps and as a result I'll vote against it." Republican Sen. Lindsey Graham of South Carolina complained that the plan reduces military benefits. 2016 in play Three leading tea party-backed senators with 2016 presidential aspirations -- Rand Paul of Kentucky, Ted Cruz of Texas, and Marco Rubio of Florida -- also have come out against the budget compromise for similar reasons. CNN's Dana Bash, Paul Steinhauser and Dan Merica contributed to this story. WASHINGTON, DC : On Monday, Senator Orrin Hatch (R-UT), pictured here in 2012, along with Senator Johnny Isakson (R-Ga.) added their names to a growing list of Republican sentators who declared their support for the bipartisan deal. (Bill O'Leary/WASHINGTON POST) Despite a concerted attack by conservative advocacy groups, a bipartisan deal to roll back sharp spending cuts known as the sequester appeared on track to clear the Senate after a growing number of Republicans declared their support for the measure. On Monday, Sens. Orrin G. Hatch (Utah) and Johnny Isakson (Ga.) added their names to a list that included Sens. John McCain (Ariz.), Susan Collins (Maine) and Ronald H. Johnson (Wis.). At least two others who were undecided on the substance of the agreement said they will nonetheless help the Senate’s 55 Democrats assemble the 60 votes needed to clear a critical procedural hurdle Tuesday morning. The federal budget accord “isn’t everything I’d hoped it would be, and it isn’t what I would have written,” Hatch said in a statement. “But sometimes the answer has to be yes.” The deal, struck by Senate Budget Committee Chairman Patty Murray (D-Wash.) and House Budget Committee Chairman Paul Ryan (R-Wis.), is intended to end nearly three years of acrimony over the budget in Washington. It would cancel half the sequester cuts for the current fiscal year, replace them with other savings and allow Congress to avert another government shutdown in January. Last week, the agreement sailed through the House on a vote of 332 to 94, raising hopes that Republicans were finally drawing to a close an era of uncompromising confrontation over the budget. But trouble quickly cropped up in the usually more moderate Senate, where a slew of tea party heroes — and GOP incumbents facing tea-party challengers — lambasted the deal. Following the lead of outside groups such as Heritage Action for America and the Club for Growth, several Republican senators complained that the agreement would allow agency spending to rise over the next two years in exchange for the vow of spending reductions nearly a decade from now. “It increases spending by $60 billion over the next two years but promises to pay for it with cuts over the next 10 [years] — something that we know Congress never gets back to actually carrying out,” said Sen. Marco Rubio (Fla.), a possible contender for the 2016 GOP presidential nomination. More worrying for advocates of the agreement, two key Republican allies in the fight against the sequester — Sens. Lindsey O. Graham (S.C.) and Kelly Ayotte (N.H.) — said late last week that they, too, would vote against the measure. Although the agreement would cancel a hit to the Pentagon budget set for January, an outcome Graham and Ayotte have been campaigning for months to prevent, the pair complained that the deal also would dial back cost-of-living increases for the youngest military retirees. “It’s unacceptable to single out our men and women in uniform in this way,” Ayotte said. Some Democrats, meanwhile, were grumbling that the deal includes no provision for extending unemployment insurance for the long-term jobless. Those benefits are set to expire at the end of December, cutting off 1.3 million people. Although senior Senate aides never doubted their ability to assemble the 50 votes needed to pass the agreement, their ability to get 60 votes to squash a threatened GOP filibuster seemed less certain. So Murray and Ryan worked through the weekend to shore up support. House Speaker John A. Boehner (R-Ohio) also reached out privately to a few of his closest friends. By Monday morning, the tide had turned, and Sen. Charles E. Schumer (N.Y.), the No. 3 Democrat in the Senate, said on MSNBC that it is “safe to say” the measure will pass the chamber. A final vote could come as soon as Tuesday evening, if Senate Republicans agree to speed things up. Otherwise, the chamber is likely to send the measure to the White House late Wednesday. The proposal would raise about $85 billion over the next decade through a variety of policies, including trimming pensions for civil-service workers and military retirees younger than 62, raising security fees for airline passengers and extending the sequester for Medicare providers an additional two years, through 2023. About $62 billion of those savings would go to replace a portion of the sequester in fiscal 2014 and 2015, restoring spending for the Pentagon and domestic agencies. The rest of the savings, about $23 billion, would go toward trimming deficits projected to exceed $6 trillion over the next decade. In announcing his support, Hatch said the agreement is “built on the necessary consensus that reflects divided government.” “I appreciate the hard work of Paul Ryan, Speaker Boehner and House Republicans in crafting this commendable compromise that reduces our debt over the long-term, prevents another government shutdown, and stops the budget battles that have rocked America with economic uncertainty and political pessimism,” Hatch said. “Much more work needs to be done.... My hope is that this budget agreement paves the way to greater stability, lasting deficit reduction, and the political will to tackle those challenges in the near future.” Republicans in the Senate could slow the progress of the bipartisan budget that passed the House last week, amid signs that a procedural vote scheduled for Tuesday may be tight. Although Democratic supporters of the deal in the Senate believe it will attract enough support to achieve the required 60 votes to pass the procedural motion, some are warning that frayed relations with Republicans could result in the numbers being tight. The $1tn deal, forged between Republican congressman Paul Ryan and Democratic senator Patty Murray, passed overwhelmingly in the House, by 332 to 94. If passed by the Senate, the agreement, which reduces blanket spending cuts and raises some new revenues without increasing taxes, would stave off the threat of another government shutdown for two years. However, the compromise does not entirely eradicate the possibility of the kind of brinksmanship which took Washington to the precipice earlier this year. While promoting his bill as a means to avoid another calamitous government closure, Ryan indicated that Republicans were still considering what they could "get out" of possible negotiations over the debt limit. “We don't want nothing out of this debt limit. We're going to decide what it is we can accomplish out of this debt-limit fight,” Ryan told Fox News Sunday. The White House has repeatedly said it will not negotiate over the debt limit, arguing that the routine increase to the Treasury's borrowing capacity should not be used as a bargaining tool. In reality, administration officials did discuss the limit during talks in October, and some Republicans may seek to use the technique to foster further concessions in an election year. For now, Ryan is focused on persuading Republicans in the Senate to support his bill and avoid a filibuster. If the bill had unanimous Democrat support, at least five Republicans would have to vote with them to move to a final vote. But if, as expected, a handful of Democrats angry that it fails to renew unemployment benefits for over 1 million Americans decided to vote against, more Republican votes will be required. Richard Durbin, the Senate majority whip, estimated that about eight Republicans were needed to support the bill. “The struggle is still on in the United States Senate,” he told CBS’s Face the Nation. “I feel we’ll have a good, strong showing from the Democratic side. But we need bipartisan support to pass it.” Conservatives have complained that the Ryan-Murray deal will increase federal spending and reduce support for working-age military veterans. Although unrelated to the budget, Republicans in the Senate are also fuming over a decision by Democrats to change the rules over the confirmation of administration appointments – robbing the Republicans of an opportunity to obstruct Barack Obama’s nominations to key posts. Democratic majority leader Harry Reid held two all-night sessions in the Senate last week, to force through Obama’s appointments. In an ambitious schedule, Reid is aiming to secure the confirmation of Jeh Johnson, Obama’s choice to replace Janet Napolitano as secretary for homeland security, and possibly Janet Yellen, who would become the first woman to head the Federal Reserve, before the Senate goes into recess on Friday. However, Reid’s focus this week will inevitably be on the budget, particularly given that so many senior Republican senators, including the minority leader Mitch McConnell and party whip John Cornyn, have signalled their opposition to the deal. Only a handful of Republicans have so far signalled that they may vote in favour of the bill, including, most recently, the Wisconsin senator Ron Johnson. The decision by Johnson, a Tea Party-aligned senator, to support the deal could be pivotal. The host of staunchly conservative groups which often steer Republican votes, such as Club For Growth and Heritage Action, lambasted the budget deal because it waters down planned spending cuts. Most Republicans in the House took the unusual step of ignoring the guidance from the rightwing groups, but Republican senators are seemingly more cautious. As Durbin pointed out, they include potential presidential hopefuls, who would want to maintain the support of the radical conservative base, as well more moderate Republicans, such as McConnell and Cornyn, facing primary challenges from Tea Party opponents in the new year. | Summary: Conservatives still don't love the bipartisan budget deal passed by the House last week, but as expected, it advanced in the Senate today after a number of Republican senators declared their support. In some cases, their quite tepid support: The deal "isn't everything I'd hoped it would be, and it isn't what I would have written," reads Orrin Hatch's statement, per the Washington Post. "But sometimes the answer has to be yes." Along with the backing of John McCain and at least five other Republicans, the Senate advanced the measure over a filibuster threshold on a 67-33 vote, the AP reports. That means it will pass the Senate no later than tomorrow and head to the White House for President Obama's signature. The bipartisan Paul Ryan-Patty Murray deal ran into trouble with the Senate despite easily passing the House, thanks to what the Post refers to as "a slew of tea party heroes" raising issues with it. Even many Democrats have problems with the bill, particularly because it doesn't renew unemployment benefits for more than a million people, CNN reports. But the deal means two blissful years of no government shutdown threats, the Guardian reports. Even so, don't get comfortable: The next battle will be over the debt ceiling, the Wall Street Journal reports. The Treasury says it should be raised by early March in order to avoid a government default, and conservatives want to try and get whatever they can in return. Then again, some think it may not be worth the potential bad press; plus, Obama has repeatedly said he won't negotiate. | 3,356 | 353 | multi_news | en |
Summarize: "Ha! ha! ha! But you know there is no such thing as choice in reality, say what you like," you will interpose with a chuckle. "Science has succeeded in so far analysing man that we know already that choice and what is called freedom of will is nothing else than--" Stay, gentlemen, I meant to begin with that myself I confess, I was rather frightened. I was just going to say that the devil only knows what choice depends on, and that perhaps that was a very good thing, but I remembered the teaching of science... and pulled myself up. And here you have begun upon it. Indeed, if there really is some day discovered a formula for all our desires and caprices--that is, an explanation of what they depend upon, by what laws they arise, how they develop, what they are aiming at in one case and in another and so on, that is a real mathematical formula--then, most likely, man will at once cease to feel desire, indeed, he will be certain to. For who would want to choose by rule? Besides, he will at once be transformed from a human being into an organ-stop or something of the sort; for what is a man without desires, without free will and without choice, if not a stop in an organ? What do you think? Let us reckon the chances--can such a thing happen or not? "H'm!" you decide. "Our choice is usually mistaken from a false view of our advantage. We sometimes choose absolute nonsense because in our foolishness we see in that nonsense the easiest means for attaining a supposed advantage. But when all that is explained and worked out on paper (which is perfectly possible, for it is contemptible and senseless to suppose that some laws of nature man will never understand), then certainly so-called desires will no longer exist. For if a desire should come into conflict with reason we shall then reason and not desire, because it will be impossible retaining our reason to be SENSELESS in our desires, and in that way knowingly act against reason and desire to injure ourselves. And as all choice and reasoning can be really calculated--because there will some day be discovered the laws of our so-called free will--so, joking apart, there may one day be something like a table constructed of them, so that we really shall choose in accordance with it. If, for instance, some day they calculate and prove to me that I made a long nose at someone because I could not help making a long nose at him and that I had to do it in that particular way, what FREEDOM is left me, especially if I am a learned man and have taken my degree somewhere? Then I should be able to calculate my whole life for thirty years beforehand. In short, if this could be arranged there would be nothing left for us to do; anyway, we should have to understand that. And, in fact, we ought unwearyingly to repeat to ourselves that at such and such a time and in such and such circumstances nature does not ask our leave; that we have got to take her as she is and not fashion her to suit our fancy, and if we really aspire to formulas and tables of rules, and well, even... to the chemical retort, there's no help for it, we must accept the retort too, or else it will be accepted without our consent...." Yes, but here I come to a stop! Gentlemen, you must excuse me for being over-philosophical; it's the result of forty years underground! Allow me to indulge my fancy. You see, gentlemen, reason is an excellent thing, there's no disputing that, but reason is nothing but reason and satisfies only the rational side of man's nature, while will is a manifestation of the whole life, that is, of the whole human life including reason and all the impulses. And although our life, in this manifestation of it, is often worthless, yet it is life and not simply extracting square roots. Here I, for instance, quite naturally want to live, in order to satisfy all my capacities for life, and not simply my capacity for reasoning, that is, not simply one twentieth of my capacity for life. What does reason know? Reason only knows what it has succeeded in learning (some things, perhaps, it will never learn; this is a poor comfort, but why not say so frankly?) and human nature acts as a whole, with everything that is in it, consciously or unconsciously, and, even if it goes wrong, it lives. I suspect, gentlemen, that you are looking at me with compassion; you tell me again that an enlightened and developed man, such, in short, as the future man will be, cannot consciously desire anything disadvantageous to himself, that that can be proved mathematically. I thoroughly agree, it can--by mathematics. But I repeat for the hundredth time, there is one case, one only, when man may consciously, purposely, desire what is injurious to himself, what is stupid, very stupid--simply in order to have the right to desire for himself even what is very stupid and not to be bound by an obligation to desire only what is sensible. Of course, this very stupid thing, this caprice of ours, may be in reality, gentlemen, more advantageous for us than anything else on earth, especially in certain cases. And in particular it may be more advantageous than any advantage even when it does us obvious harm, and contradicts the soundest conclusions of our reason concerning our advantage--for in any circumstances it preserves for us what is most precious and most important--that is, our personality, our individuality. Some, you see, maintain that this really is the most precious thing for mankind; choice can, of course, if it chooses, be in agreement with reason; and especially if this be not abused but kept within bounds. It is profitable and sometimes even praiseworthy. But very often, and even most often, choice is utterly and stubbornly opposed to reason... and... and... do you know that that, too, is profitable, sometimes even praiseworthy? Gentlemen, let us suppose that man is not stupid. (Indeed one cannot refuse to suppose that, if only from the one consideration, that, if man is stupid, then who is wise?) But if he is not stupid, he is monstrously ungrateful! Phenomenally ungrateful. In fact, I believe that the best definition of man is the ungrateful biped. But that is not all, that is not his worst defect; his worst defect is his perpetual moral obliquity, perpetual--from the days of the Flood to the Schleswig-Holstein period. Moral obliquity and consequently lack of good sense; for it has long been accepted that lack of good sense is due to no other cause than moral obliquity. Put it to the test and cast your eyes upon the history of mankind. What will you see? Is it a grand spectacle? Grand, if you like. Take the Colossus of Rhodes, for instance, that's worth something. With good reason Mr. Anaevsky testifies of it that some say that it is the work of man's hands, while others maintain that it has been created by nature herself. Is it many-coloured? May be it is many-coloured, too: if one takes the dress uniforms, military and civilian, of all peoples in all ages--that alone is worth something, and if you take the undress uniforms you will never get to the end of it; no historian would be equal to the job. Is it monotonous? May be it's monotonous too: it's fighting and fighting; they are fighting now, they fought first and they fought last--you will admit, that it is almost too monotonous. In short, one may say anything about the history of the world--anything that might enter the most disordered imagination. The only thing one can't say is that it's rational. The very word sticks in one's throat. And, indeed, this is the odd thing that is continually happening: there are continually turning up in life moral and rational persons, sages and lovers of humanity who make it their object to live all their lives as morally and rationally as possible, to be, so to speak, a light to their neighbours simply in order to show them that it is possible to live morally and rationally in this world. And yet we all know that those very people sooner or later have been false to themselves, playing some queer trick, often a most unseemly one. Now I ask you: what can be expected of man since he is a being endowed with strange qualities? Shower upon him every earthly blessing, drown him in a sea of happiness, so that nothing but bubbles of bliss can be seen on the surface; give him economic prosperity, such that he should have nothing else to do but sleep, eat cakes and busy himself with the continuation of his species, and even then out of sheer ingratitude, sheer spite, man would play you some nasty trick. He would even risk his cakes and would deliberately desire the most fatal rubbish, the most uneconomical absurdity, simply to introduce into all this positive good sense his fatal fantastic element. It is just his fantastic dreams, his vulgar folly that he will desire to retain, simply in order to prove to himself--as though that were so necessary--that men still are men and not the keys of a piano, which the laws of nature threaten to control so completely that soon one will be able to desire nothing but by the calendar. And that is not all: even if man really were nothing but a piano-key, even if this were proved to him by natural science and mathematics, even then he would not become reasonable, but would purposely do something perverse out of simple ingratitude, simply to gain his point. And if he does not find means he will contrive destruction and chaos, will contrive sufferings of all sorts, only to gain his point! He will launch a curse upon the world, and as only man can curse (it is his privilege, the primary distinction between him and other animals), may be by his curse alone he will attain his object--that is, convince himself that he is a man and not a piano-key! If you say that all this, too, can be calculated and tabulated--chaos and darkness and curses, so that the mere possibility of calculating it all beforehand would stop it all, and reason would reassert itself, then man would purposely go mad in order to be rid of reason and gain his point! I believe in it, I answer for it, for the whole work of man really seems to consist in nothing but proving to himself every minute that he is a man and not a piano-key! It may be at the cost of his skin, it may be by cannibalism! And this being so, can one help being tempted to rejoice that it has not yet come off, and that desire still depends on something we don't know? You will scream at me (that is, if you condescend to do so) that no one is touching my free will, that all they are concerned with is that my will should of itself, of its own free will, coincide with my own normal interests, with the laws of nature and arithmetic. Good heavens, gentlemen, what sort of free will is left when we come to tabulation and arithmetic, when it will all be a case of twice two make four? Twice two makes four without my will. As if free will meant that! | Summary: The Underground Man again provides a retort on behalf of his readers, who might suppose that science makes that whole idea of "choice" into a contradiction. Now we're back to this idea of a big giant formula: if we knew enough about science, we could predict every action, feeling, and event. But, he posits, if such a formula is created to predict man's desires, man will intentionally stop desiring things. Now he speaks on our behalf again: surely, we are thinking that if reason and desire went up against each other in a kind of Mortal Combat-style death match, reason would win. And because choice and reason can be calculated, this big giant formula will someday be determined. And then, the next thirty or however many years of our lives will be figured out before we live them. But as difficult as that might be to accept, surely we have to accept it, because nature is nature and we can't change it. And that's where the Underground Man begs to differ. Reason isn't everything, he says. Reason only satisfies half of man. Human nature, he says, is made of both reason and impulses. So if we want to argue that man will always act advantageously, on the grounds that it can be proven mathematically, that's fine; it CAN be proven - but only mathematically, and that's just not sufficient, since mathematics are only part of man. It follows then, that often times, doing stupid things actually are to our advantage. The advantage of free will, he explains, is that it preserves that which is most important to us: our individuality. What we choose is sometimes in agreement with reason, but very often it is not. Now, says the Underground Man, let's suppose for a moment that man is not stupid. If man isn't stupid, the Underground Man argues, then he is at least ungrateful. In fact, that's how we should define man: the ungrateful biped. But being ungrateful isn't even his worst feature: his "moral obliquity" is. His WHAT? Yes, the Underground Man says, this moral obliquity causes man's second-worst feature: his lack of good sense. And man has been acting this way since, well, forever. He then decides to look briefly at the history of the universe. Really briefly. Thinking about the large bronze statue of Helios in Rhodes, he concludes that the history of mankind is majestic. Thinking about the many different uniforms worn by many different armies of soldiers, he concludes that the world is multi-colored. He continues in this vein, proving that you can say anything you want, really, about the history of the universe and then justify it with some example. There is one description, however, which you cannot justify: you cannot say that the world is rational. Despite this, moral and rational men keep popping up everywhere, like so many whack-a-moles. But the Underground Man maintains his argument: all the moral men will ultimately do something immoral, and all the rational men will eventually do something stupid. So what would happen, asks the Underground Man, if you gave man everything he wanted? Even cake? In that case, he argues, man would risk losing all that yummy cake and seek out misery and act in "vulgar folly" just to prove that he can. This is what the Underground Man means when he talks about ingratitude; man is ungrateful for his tasty cake. AND, if he can't easily achieve his misery, he will cause destruction and chaos for the entire universe in order to have it. This, the Underground Man notes, is the main difference between man and animals: only man can launch such a curse and destruction upon the world. But, we might say, the big giant scientific formula would know all this ahead of time by calculating it through reason. In that case, he says, man would just go crazy in order to be rid of reason once and for all. And what if we argue that free will just happens to coincide with the laws of nature, but that the coincidence doesn't make it any less free? Well...that's just not true, says the Underground Man. Twice two makes four whether we will it to or not. So how can our will be free? It can't, at least not when we're living according to the laws of nature. | 2,566 | 961 | booksum | en |
Write a title and summarize: Amebiasis is a protozoal infection caused by Entamoeba histolytica, while the morphologically indistinguishable E. dispar is considered as non-pathogenic. Polymerase chain reaction (PCR) assays are necessary to differentiate both species. The most common clinical presentations of E. histolytica disease are amebic colitis and amebic liver abscess, but asymptomatic infection is also possible. We assessed the frequency and pattern of clinical symptoms and microscopic features in travelers/migrants associated with E. histolytica intestinal infection and compared them to those found in individuals with E. dispar infection. We conducted a retrospective study at the travel clinic of the Institute of Tropical Medicine, Antwerp, Belgium on travelers/migrants found from 2006 to 2016 positive for Entamoeba histolytica/dispar through antigen detection and/or through microscopy confirmed by PCR. All files of individuals with a positive PCR for E. histolytica (= cases) and a random selection of an equal number of Entamoeba dispar carriers (= controls) were reviewed. We calculated the sensitivity, specificity and likelihood ratios (LRs) of clinical symptoms (blood in stool, mucus in stool, watery diarrhea, abdominal cramps, fever or any of these 5 symptoms) and of microscopic features (presence of trophozoites in direct and in sodium acetate-acetic acid-formalin (SAF) -fixed stool smears) to discriminate between E. histolytica and E. dispar infection. Of all stool samples positive for Entamoeba histolytica/dispar for which PCR was performed (n = 810), 30 (3. 7%) were true E. histolytica infections, of which 39% were asymptomatic. Sensitivity, specificity and positive LRs were 30%, 100% and 300 (p 0. 007) for presence of blood in stool; 22%, 100% and 222 (p 0. 03) for mucus in stool; 44%, 90% and 4. 7 (p 0. 009) for cramps and 14%, 97% and 4. 8 (p = 0. 02) for trophozoites in direct smears. For watery diarrhea, fever and for trophozoites in SAF fixated smears results were non-significant. E. histolytica infection was demonstrated in a small proportion of travelers/migrants with evidence of Entamoeba histolytica/dispar infection. In this group, history of blood and mucus in stool and cramps had good to strong confirming power (LR+) for actual E. histolytica infection. Trophozoites were also predictive for true E. histolytica infection but in direct smears only. Amebiasis is a protozoal infection caused by Entamoeba histolytica. The most common clinical presentations of disease are amebic colitis and amebic liver abscess. Before molecular tests allowed distinction between Entamoeba species[1], [2], the estimations of the worldwide burden of amoebiasis indicated that approximately 500 million people were infected by E. histolytica, and 10% of these individuals had invasive amoebiasis. Moreover, it was estimated that 100,000 patients per year died due to the clinical complications of the disease[3]. The genus Entamoeba contains many species of which Entamoeba histolytica, Entamoeba dispar, Entamoeba coli, Entamoeba hartmanni, and to a much lesser extent Entamoeba moshkovskii and Entamoeba polecki, are found in the human intestinal tract. Cysts of E. histolytica, E. dispar, and E. moshkovskii are morphologically indistinguishable[4], [5], [6] but the species are biochemically and genetically different[7]. Towards the end of the 20th century, Polymerase Chain Reaction (PCR) -assays that allowed to differentiate between E. histolytica and E. dispar infection led to a re-assessment of the disease burden and indicate that earlier reports had largely overestimated the true number of E. histolytica infections. More recent reports showed in addition varied frequencies of asymptomatic E. histolytica carriage in different populations, ranging from 0–2% in South-Africa and Ivory Coast to 21% in Egypt, with intermediate prevalence of 13. 8% reported in rural Mexico and 9. 6% in Vietnam[8], [9], [10], [11]. In studies dating from before PCR could discriminate between E. histolytica and E. dispar infection, a 4% prevalence of asymptomatic E. histolytica/dispar infection was found in travelers returning from the tropics[12]. Notwithstanding, the ratio of symptomatic vs asymptomatic E. histolytica infections remains largely unknown. Though E. dispar is considered non-pathogenic, it has been reported that E. dispar may be the causative agent of intestinal and extra-intestinal symptoms in humans[13], [14]. The finding of trophozoites (or vegetative forms) in fresh stool samples is generally considered predictive of true E. histolytica infection, especially when large trophozoites containing red blood cells are found (hematophagy) [15], [16], [17], but it is not known whether the presence of trophozoites found after fixation of stools differs between E. histolytica and E. dispar. In the present work, we aimed to determine the frequency of E. histolytica infection among travelers and migrants presenting with an Entamoeba histolytica/dispar infection diagnosed by microcopy and/or antigen detection at the travel clinic of the Institute of Tropical Medicine of Antwerp, Belgium. In addition, we assessed the predictive value of microscopic features and clinical symptoms for E. histolytica intestinal infection in this study group and correlated the finding of trophozoites in fresh and fixed stool samples with species identification. The Institute of Tropical Medicine, Antwerp (ITMA) is the national reference clinic for tropical medicine in Belgium, with on average about 6500 consultations a year for post-travel care. For this retrospective study, all files of symptomatic and asymptomatic individuals having attended the travel clinic of the ITMA from May 2006 to March 2016 and positive for Entamoeba histolytica/dispar through antigen detection and/or through microscopy (trophozoites or cysts) confirmed by PCR, were retrieved. The medical records of all travelers and migrants proven to be infected with E. histolytica during the study period were then reviewed. An equal number of files of patients with confirmed E. dispar intestinal infection were randomly chosen and analyzed for a case control comparison. Relevant clinical and laboratory data were extracted, de-identified and entered in a Microsoft Access 2010 database. Variables included: demographic data including country of origin, month and year of first Entamoeba positive test, most recent travel destination and, for the symptomatic included cases and controls, the following clinical features at presentation: blood in stool, mucus in stool, watery diarrhea, abdominal cramps and fever, as reported in the medical files. All stool samples were analyzed by microscopic examination of direct smears and wet mounts after formalin-ether concentration (Loughlin and Spitz, 1949[18]). A limited number of samples with high suspicion for amebic dysentery was urgently sent to the lab for immediate examination. In case a fresh stool sample could not be produced in ITMA, the patient received a package to collect stools at home and instructions to mix part of the stools immediately with a sodium acetate-acetic acid-formalin (SAF) solution. Both fixed and unfixed portions were sent to ITMA for examination. In case the stool sample was produced at ITMA, part of it was mixed with SAF-solution within 20 minutes on request by the treating physician. All SAF-fixed stool samples were examined by microscopy after iron hematoxylin Kinyoun staining. Antigen detection with the enzyme-linked immunosorbent assay (ELISA) E. histolytica ProSpecT ELISA Microplate assay (Remel, Lenexa, Kansas, USA), was performed when requested by the treating physician. Since microscopic distinction of E. histolytica, E. dispar and some other Entamoeba species is not possible, an E. histolytica and E. dispar specific real-time PCR (Cnops and Van Esbroeck, 2010[19]) was performed on all samples positive by microscopy and/or antigen detection. Direct smears were examined for the presence of hematophagy. In SAF-fixed stool this feature cannot be used, given possible superposition of erythrocytes over parasites, instead of within parasites. Among individuals found with E. histolytica/dispar intestinal infection, we analyzed the respective frequencies of the presence of E. histolytica and E. dispar trophozoites and cysts as well as the pattern of clinical findings (blood and/or mucus in stool, watery diarrhea, presence of abdominal cramps, fever or any symptom). Sensitivity, specificity and likelihood ratios (LRs) were calculated, using the PCR as reference diagnostic standard. Finally, we assessed whether hematophagy can be used as a criterium to distinguish E. histolytica and E. dispar species in direct stool smears. Laboratory test results were stored in the Laboratory Information System AS/400 (IBM, USA). Data mining was performed with the SAP Business Objects (SAP, USA) program. Statistical analyses were done with Epi-Info (CDC 2015). Dichotomic variables where compared with Fisher exact test, minimum significance p<0. 05. This was a retrospective analysis of data collected during clinical care over an 11-year period. Ethical clearance was obtained from the institutional review board at ITMA. Laboratory queries were obtained in an anonymous way. Clinical data were then retrieved through an encoded link and de-identified for analysis according to the Belgian legislation. From May 2006 till March 2016 parasitological examination was performed on 40,638 stool samples. Of these 868 (2. 1%) were found positive for Entamoeba histolytica/dispar through antigen detection and/or through microscopy confirmed by PCR. After removing results of follow-up samples, E. histolytica was detected in 30/826 samples: 3. 6% of all stool samples positive for E. histolytica/dispar and 0. 07% of all examined stool samples. E. dispar was detected in 714 (86. 4%) samples, neither E. histolytica nor E. dispar in 50, and PCR was technically not feasible in 16 because no fresh stool sample was received. No co-infections with E. histolytica and E. dispar were found. Antigen detection was performed in 396 of the 744 samples with E. histolytica or E. dispar as confirmed by PCR. In 16 samples, the antigen test was positive, with negative PCR for E. histolytica or E. dispar and negative microscopy (or microscopy not done), while in 1 E. histolytica PCR-confirmed patient antigen testing was positive with negative microscopy. The antigen test was positive in 15/16 (94%) E. histolytica positive and 275/380 (72%) E. dispar positive samples. When only examination of direct smears was considered, the finding of trophozoites was predictive of E. histolytica (p = 0. 02), although sensitivity was very low (14%) (Table 3) In contrast, the finding of trophozoites in fixed samples was not predictive of E. histolytica (p = 0. 2; Table 4). In our Belgian reference clinic for tropical medicine we identified 3. 6% (30/826) of Entamoeba histolytica/dispar infections as true E. histolytica infections by PCR. This confirms the finding in other studies[20], [21] that the bulk of Entamoeba histolytica/dispar infections are caused by E. dispar amoeba. True E. histolytica enteritis is a rare finding in patients presenting in our reference center, with on average less than 3 cases detected per year. In our study, the presence of blood or mucus in stool or abdominal cramps are clearly significant predictors (p < 0. 005) of true E. histolytica infections in case Entamoeba histolytica/dispar cysts or trophozoites were found on microscopy. Likelihood ratios of symptoms can be used similarly to test results to calculate the probability of disease according to the Bayes theorem. Since the positive LR is the ratio between true positive and false positive rates, a symptom, even if infrequent in a given disease, can have a high LR+ (a high confirming power) if it is rarer in the competing[22]. Indeed we observed that blood or mucus in stool or abdominal cramps were not that frequent in true E. histolytica infections, but that these symptoms were almost never present in the matched patients with E. dispar, which explains the high LR+. Therefore, in a context where only microscopy is available, a patient presenting with blood or mucus in stool or cramps should anyhow be treated as amoebiasis if Entamoeba histolytica/dispar cysts/trophozoites are found. Nevertheless it is also worth noting that a sizeable proportion of E. histolytica cases were asymptomatic. Relying only on one of the three clinical predictors would have missed 10 true E. histolytica infections in our cohort. Hematophagy is considered a discriminative microscopic criterion to distinguish E. histolytica from E. dispar infection[15], [16], [17]. This was also demonstrated in this study in which 5/5 hematophagous trophozoites found in immediately examined samples proved to be E. histolytica. Finding trophozoites in direct smears had a LR+ for E. histolytica of 4. 8, corresponding to a good confirming power. However, the LR- of 0. 9 indicated that the absence of trophozoites, did not rule out E. histolytica infection. The non-significant LR+ of 1. 2 for trophozoites in SAF fixed stool samples confirmed that this method cannot be used for species prediction. The non-pathogenicity of Entamoeba dispar is questioned by several authors[23], [14]. A study by Ximénez and colleagues suggests the existence of several different genotypes of E. dispar that can be associated to, or be potentiality responsible for, intestinal or liver tissue damage, similar to that observed with E. histolytica[13]. The difference in percentage of patients presenting with any symptom in patients with mono-infections with E. histolytica vs E. dispar was not significant (61% vs 55%, p value 0. 42). This is not equivalent to stating that all symptoms of the 55% patients with symptomatic E. dispar infections were attributable to the E. dispar amoebae. Our study was not designed to show a pathogenic effect of E. dispar. However, the high frequency of symptoms in patients with E. dispar mono-infection supports Ximénez’s hypothesis, but symptoms in E. histolytica infected patients were clearly more often suggestive of intestinal tissue invasion. Our study has several limitations. It was a single-center study and the total number of E. histolytica infections found might not be representative for all returning travelers. In patients consulting at our center, we found 30 E. histolytica infections over 10 years, whereas the total number of E. histolytica infections diagnosed in our laboratory receiving stool samples from all over Belgium was 124 over the same period. Next, it was a retrospective study meaning that collection of data was not systematic. However, given the low number of confirmed E. histolytica infections in the 810 samples tested by PCR, the impact of missing analyses is likely marginal. In 50 samples positive by microscopy PCR was negative for both E. histolytica and E. dispar which probably indicates incorrect identification as infections with species such as E. moshkovskii and E. polecki are considered to be rare. A difference in clinical presentation in patients with E. histolytica and E. dispar infection is a possible confounding factor since clinicians might have asked less stool samples in asymptomatic patients. This might have underestimated the true prevalence of these infections. Nevertheless, the proportion of asymptomatic patients in our case-control group did not differ significantly. Furthermore, requesting stool analysis including antigen testing was clinician driven and an unknown number of E. histolytica/dispar infections may have been missed, in particular in asymptomatic travelers. The most trustworthy method to detect all E. histolytica and E. dispar infections, would have been to perform PCR on all stool samples of all symptomatic and asymptomatic travelers[7], [24]. During the study period, this method was not part of common practice, though this may change with the deployment of multiplex PCR platforms to analyze stool samples. Last, quantification of pathogens is usually linked with disease severity, which is mostly demonstrated for bacterial diseases[25]. We opted however to correlate our symptoms to the qualitative and not the quantitative interpretation of the PCR results because the goal of our study was identification of E. histolytica as such–which is treated even in asymptomatic patients–and not determination of pathogenicity. In conclusion, even in a national reference travel clinic in Europe, E. histolytica intestinal infections are rarely diagnosed. Finding trophozoites is helpful in discriminating between E. histolytica and E. dispar infection in direct smears but not in SAF fixed samples. History of blood and mucus in stool and cramps in individuals with microscopic evidence of E. histolytica/dispar infection had good to strong predictive weights for actual E. histolytica infection. Hematophagy was a very rare finding but in our experience was always associated, when requested, with E. histolytica infection. Our study suggests that E. dispar might be pathogenic but symptoms in E. histolytica infected patients were clearly more often suggestive of intestinal tissue invasion. | Title: Clinical and microscopic predictors of Entamoeba histolytica intestinal infection in travelers and migrants diagnosed with Entamoeba histolytica/dispar infection Summary: In the present work, we found that E. histolytica intestinal infections are rarely diagnosed among travelers and migrants presenting in a national reference travel clinic in Europe. Microscopic finding of cysts or trophozoites and antigen testing cannot discriminate between Entamoeba histolytica/dispar infection, which leads to overdiagnosis of E. histolytica infections in low resource settings where PCR is not available. We found visualization of trophozoites under the microscope helpful in discriminating between E. histolytica and E. dispar infection in direct smears. Hematophagy is a very rare finding but in our experience was always associated with E. histolytica infection. In a context where only microscopy is available, a patient presenting with blood or mucus in stool or cramps should anyhow be treated as amoebiasis if Entamoeba histolytica/dispar cysts/trophozoites are found. Nevertheless it is worth noting that a sizeable proportion of E. histolytica cases were asymptomatic. Last, our study suggests that E. dispar might be pathogenic but symptoms in E. histolytica infected patients were clearly more often suggestive of intestinal tissue invasion. | 4,305 | 300 | lay_plos | en |
Summarize: Published on Mar 2, 2017 Zeus Campbell Aka Top Rope Zeus Is the actor in the New Bat tax commercial and House Republicans in Congress have proposed the Border Adjustment Tax, also known as the B.A.T. This would be a new, additional tax on the products Americans use every day. This destructive tax increase is a "job-killing formula" that's bad for consumers and bad for retailers. NRF's new "As Seen on TV" campaign is placing this message front and center. Visit http://bat.tax to learn more about the campaign. SUBSCRIBE …ITS FREE - http://www.youtube.com/user/topropeze... NEWEST Season of Hump Day Romance - https://www.youtube.com/watch?v=5iCj_... SEASON 1 -https://www.youtube.com/watch?v=ln9aI... MORE RELATIONSHIP ADVICE - https://www.youtube.com/watch?v=Pdxv6... STALK ME FB - https://www.facebook.com/zeustoprope/ IG - @TopRopeZeus TWITTER - https://twitter.com/TopRopeZeus Related Topics that Top Rope Zeus videos Addresses : Dating Advice For Women,Relationship Advice For Women,Relationship Coach For Women, Dating Coach For Women Dating, Relationships, understanding men, Dating Advice, Love Advice Relationship Advice, How Men Think, What Men Want, What attracts men, How to attract a man, how to create lasting love, how to know if he likes you, signs your man likes you.Affairs, infidelity/ cheating this includes infidelity, one night stands, internet relationships (including sexting),long and short term affairs and financial infidelity.Top rope Zeus talks give advice on Sexual problems,life stages, core values and beliefs. Sometimes people out grow each other, stress, work related stress, bored in your relationships, jealous partner, blended family issues. Domestic violence should not have gotten married, divorce, unrealistic expectations and social media messing up relationships. Top Rope Zeus videos also deal with disciplines perceived lack of concern,care, consideration. Your partner is In prison well I don't have a lot of experience in that one lolStay In Your Lane Insurance is one of Top Rope Zeus Biggest episodes of Hump Day Romance forcing People to Stay In There LANE! …Top Rope Zeus Also brought you funny videos like “ Woman Gives Birth during Super bowl, Stay in your lane Insurance, Chris Brown Parody, Where the Fuck is Your Hair aka The Natural Hair Song, Worlds Most Honest Wedding vows and of course the Jidenna Classic Man Parody. Don't Forget to watch Stay In Your Lane Insurance 2! MORE FUNNY VIDEOS - https://www.youtube.com/watch?v=5iCj_... 1. Stay In Your Lane Insurance 2- [ WOMEN'S Edition ] - https://www.youtube.com/watch?v=5iCj_... 2. WHERE THE FUCK IS YOUR HAIR - https://www.youtube.com/watch?v=SzCwr... 3. WOMAN GIVE BIRTH DURING SUPER BOWL -https://www.youtube.com/user/TopRopeZ... 4. [BANNED] BEATS BY DRE COMMERCIAL- https://www.youtube.com/watch?v=ln9aI... 5. THOT POCKET [Hot Pocket Parody] - https://www.youtube.com/watch?v=QbuhA... 6. JIDENNA “ Classic Man” PARODY - https://www.youtube.com/watch?v=tTs8R... BAT fans—most notably House Speaker Paul Ryan and Ways and Means Chairman Kevin Brady—pitch the provision as an economically elegant twofer: an America-First measure that discourages companies from moving operations overseas while creating a revenue stream ($1 trillion every decade or so) that allows the overall corporate tax rate to be slashed. Opponents—most vocally Senators David Perdue and Tom Cotton—argue that a BAT is another grubby government cash grab that will ultimately hurt consumers when, say, Walmart has to jack up the prices of underwear, bananas, and Playstations. In a February 8 letter to colleagues, Perdue, who spent four decades in the business world, charged that the BAT is “regressive, hammers consumers, and shuts down economic growth.” Thus the battle lines are drawn. And, make no mistake, this will not be some bush-league, penny-ante skirmish. Behind the legislative factions are amassing some of the heaviest hitters in corporate America, ready to spend millions to sway debate on behalf of their team. Roughly speaking, companies that do a lot of exporting dig the BAT (think: Boeing, Merck, and Dow Chemical) while import-dependent retailers (including Target, Nike, and, yes, Walmart) fear it will destroy their bottom lines. The oil industry isn’t feeling much BAT love either. The Koch brothers want it dead, like, yesterday. At this point, anti-BATers have an edge. Why? Partly, because the provision is super complicated and almost impossible to explain in terms that don’t sound like something a coven of economists vomited up. Ask BAT fans why the provision won’t, in fact, hurt retailers or consumers, and you’re instantly hip-deep in talk of currency revaluation, purchasing power, and territorial taxation. Last Wednesday, one day after Paul Ryan tried to educate Senate Republicans on the wonders of BAT at their weekly policy lunch, Tom Cotton (who represents Walmart’s home state of Arkansas) snarked on the Senate floor, “Some ideas are so stupid only an intellectual could believe them.” This is in no way to suggest that the pro-BAT arguments are wrong. They simply don’t push the same buttons as anti-BAT warnings that Congress is poised to screw consumers in order to fund big tax cuts for corporations. For the past few weeks, in fact, an anti-BAT coalition called Americans for Affordable Products has been busy hawking this exact message. “This is a consumer tax—a means by which House Republicans are paying for other tax deductions,” asserted AAP member Brian Dodge. “It’s not about America First. It’s not a trade-deficit reduction tool. It is a pay-for.” AAP is lobbying lawmakers and staffers and doing public outreach. Last Wednesday, it dispatched eight CEOs to chat with Trump and Vice President Pence. “We view our job as leading a large education campaign,” said Dodge. “We believe the more that lawmakers understand about this proposal, the less inclined they’ll be to support it.” Canadian Prime Minister Justin Trudeau on Thursday repeated his opposition to a U.S. proposal for an import tax, telling energy executives gathered in Houston a levy would hurt both economies. “Anything that creates impediments at the border, extra tariffs, new taxes is something we’re concerned with,” Trudeau said, adding: “You’re going to be hurting not just the Canadian economy but the American economy as well.” Republicans have proposed a border adjustment tax that favors exports over imports as part of a plan to overhaul the U.S. tax code. The idea is under attack from import-heavy businesses but it is supported by large exporters, such as manufacturers. Earlier, Canada’s Natural Resources minister said his government is ready to discuss changes to the North American Free Trade Agreement (NAFTA) with the United States and Mexico. Minister Jim Carr said Ottawa’s view is that trade agreements “every now and then should be refreshed,” responding to questions about the trade pact at a news briefing in Houston. U.S. President Donald Trump has said he wants to renegotiate the more than two decade old agreement. Carr also said Canada would be prepared to respond to the U.S. administration’s border tax proposal, but said executives attending the CERAWeek energy conference told him they don’t see the need for a tax on energy imports. “They want a free movement of goods and services. We have to do a better job as Canadians of reminding our American friends how integrated this relationship is,” Carr said. He declined to speculate on what policy changes might come out of the new U.S. administration, but said that Canada would “expand and deepen the network of people we do business with in the United States.” Donald Trump and Paul Ryan Alex Wong/Getty Images The AHCA is dead, long live the AHCA. The healthcare bill's swift death occured, in part, because House Speaker Paul Ryan and the Trump administration are anxious to get to some legislative policy where they can rack up a win — specifically, tax reform. But it's not going to be that easy, especially not without the spending cuts the passage of the AHCA would've provided. Republicans needed those cuts because they are trying to pass tax reform through a mechanism called budget reconciliation. That allows a measure to be passed by a simple majority — so no Democracts can filibuster — as long as its revenue neutral (doesn't add to the deficit). Now, without the AHCA cuts, it's going to be harder to go with the substantial tax cuts Trump and Ryan's plan have proposed without adding to the deficit, so that's one hurdle. Another is in the details of Ryan's "Better Way" bill itself, and they aren't small details. Ryan's plan fundamentally changes America's tax system by significantly lowering the corporate tax rate from 35% to 20%, cutting out breaks for corporate America — like net interest deductions — and implementing a border adjustment tax (BAT). The border adjustment tax would be a huge change for us. Most simply, it's a tax on imported goods and a tax break for exported goods. The way supporters of the measure reason it, the tax domestic importers have to pay will be offset by a strengthening dollar. Plus, it will go a long way in raising revenue to make this tax bill passable through reconciliation, so fiscal hawks (like the intractable Freedom Caucus) like it. But the measure is controversial, and here's why. Domestic importers hate it. Wal-Mart and the Koch network have already mounted an offense against the legislation. The CEO of Kohl's said the impact on the consumer "would be massive" in an interview with CNBC because prices will go up dramatically. It could also result in job losses for those companies. We should note that smaller importers are even more terrified than the big guys. Republicans have said they will try to find a way to make this policy work for them, but have also said they have no plans to create an exemption for small businesses. A bunch of economists don't think the dollar will get strong enough to offset taxes on imports, turning the whole policy into a drag on the economy. It doesn't sound like the White House is totally sold on it. Trump said he didn't like it in an interview with the Wall Street Journal last month (though he's also said positive things about the proposal). And former Goldman Sachs COO and Trump's National Economic Council head Gary Cohn has sounded pretty non-commital about the whole idea. Then there's the idea that BAT could be a violation of World Trade Organization rules. Specifically, "like" goods made domestically should be treated the same (tax-wise) as goods made abroad and imported. "Publicly available descriptions of the Republican proposal raise concerns that it would permit certain deductions (and thus establish lower tax rates) for domestically-produced goods, while denying the same deductions for the same imported products," write tax experts at law firm White and Case. So, to review, the White House and Speaker Ryan are now in a position where, more than ever, they need to get the entire party on board with a very controversial policy in order to pass tax reform. Besides being untested, the policy has powerful enemies across the country, and it could also really upset our trading partners. Or as analysts at Morgan Stanley put it earlier this month: A 'border tax' in its various potential forms is the critical uncertainty in tax reform, often the differentiator between more and less positive risk outcomes. Building on the unintended consequences we've previously identified (see Reality Bites), our economists argue there isn't a clear case for the near-term benefits of a border tax,even when paired with regulatory reform,given the path for USD seen by our FX strategists. Furthermore, our corporate credit team sees meaningful risks from this provision,given elevated business model uncertainty and a weak starting point on corporate fundamentals. The German Economy Minister warned Washington against unleashing a trade war, saying Berlin would take legal action before the World Trade Organization (WTO) if it were to follow through on its import tax plans. "I'm betting partly on reason and partly on the courts" to prevent a damaging trade war, Zypries told Deutschlandfunk public radio on Friday. The warning came as Republicans in Congress are pushing for a major shake-up of the US corporate tax system that would include a new "border adjustment” system. It would see US imports subject to a tax of reportedly 20 percent and export revenues exempted. If the US border tax was found to breach WTO rules, "it wouldn't be the first time that Mr Trump has failed before the courts," Zypries said, in a jab at repeated rejections by US judges of executive orders banning immigration from majority-Muslim countries. Germany hard hit In 2016, the United States was Germany's biggest export customer, importing 107 billion euros ($115 billion) of goods while selling back just 58 billion euros' worth. During the US election campaign Trump targeted the German luxury carmaker BMW by name with threats of a border levy if it goes ahead with construction of a plant in Mexico. The new US president has also singled out exporting nations Germany and China for unfair trade policies, accusing them of manipulating their currencies to make their goods less expensive. But Berlin has repeatedly said that German products were simply better and that the US should focus on producing more competitive manufacturers. However, Zypries also acknowledged that Germany's huge trade surplus with the US was a "problem," adding that Berlin was acting to reduce it. She also stressed that "Americans need our machines and industrial plants" for the time being. Her prediction was, she said, that Trump's advisors would warn him that "the Americans would be cutting off their nose to spite their face if they slap such taxes on imports." Gearing up for WTO challenge The EU and other US trading partners have begun laying the groundwork for a legal challenge to a US border tax proposal in a move that could trigger the biggest case in WTO history. The Trump administration has criticized WTO rules that allow countries with VAT-based tax systems to offer rebates on exports while income-based systems such as the US's cannot do the same. "The unequal treatment of the US income tax system under biased WTO rules is a grossly unfair subsidy to foreigners exporting to the US and a backdoor tariff on American exports to the world that kills American jobs and drives American factories offshore," White House top trade advisor Peter Navarro said recently. Chad Bown, an expert on WTO trade disputes at the Peterson Institute for International Economics, has estimated that a US defeat before the WTO could unleash trade retaliation by other countries worth $385 billion a year. That would be almost 100 times greater than the largest WTO finding to date. Should the US ignore a WTO ruling, as Donald Trump has threatened in the past, it could lead to the unraveling of the international system designed to prevent trade wars, Bown added. Watch video 01:37 Share Mnuchin, Schäuble play down trade tensions Send Facebook Google+ Whatsapp Tumblr linkedin stumble Digg reddit Newsvine Permalink http://p.dw.com/p/2ZM32 Mnuchin, Schäuble play down trade tensions uhe/kd (Reuters, dpa) The House Republican tax plan proposes to transform the corporate income tax into a destination-based cash flow tax (DBCFT), which would include border adjustments that exempt exports but include imports in tax bills. The effects of the border adjustment on the economy depend on how exchange rates respond. However, the potential response is a source of significant confusion and uncertainty. Here’s what we do and don’t know. First, some background: The DBCFT is a modified version of a value added tax (VAT); it allows deduction for wages, while VATs do not. The VAT is a consumption tax, so it exempts the normal return from investment from tax. Thus, the DBCFT only taxes consumption financed by certain types of capital income – namely, returns to previous investments and supra normal returns to new investments. A border adjustment makes sense for a domestic consumption tax like a VAT since exports are not consumed domestically, but imported goods and services are. This makes the border adjustment a policy that levels the playing field so that all goods consumed in the US face the same tax rate regardless of where they are produced. Thus, it is quite different from a selective tariff. All advanced countries except the US already have VATs (on top of corporate income taxes), and all of those VATs are border-adjusted. The border adjustment for VATs is explicit – taxes on production are rebated for exports; imports are taxed. While explicit border adjustments may seem like a strange concept to Americans, implicit border adjustments are already present in state-level retail sales taxes. Goods produced in state A and exported to and sold in state B do not face state A’s sales tax. Goods produced in state B and imported to state A do face state A’s sales tax. The border adjustment for a cash flow tax would also be implicit – the tax would exclude exports from tax and would not allow deductions for the cost of imported goods. By ignoring foreign transactions, the cash flow tax with border adjustment also removes the incentive for firms to relocate profits or move profitable activities outside of the country. However, the border adjustment in the House Republican plan is probably not WTO-compatible. The WTO requires that imports and domestically produced goods be treated the same. But the DBCFT taxes the whole value of imports while only taxing the part of domestically produced goods that relates to above-normal returns to capital owners. This could be addressed by separating the DBCFT into a regular value-added tax and a wage subsidy elsewhere in the system. WTO issues aside, simple economic theory implies that the exchange rate should rise immediately (the dollar should go up in value) by the full extent of the tax. There are two ways to see this. First, the exemption of exports would raise demand for US dollars as foreigners need more dollars to purchase more exported US goods. Similarly, the tax on imports would restrict the supply of dollars worldwide as Americans made fewer purchases from abroad. The two effects would serve to raise the value of the dollar and have offsetting effects on the quantity of dollars traded. A second way to see this point is to note the national income accounting identity in which the difference between domestic saving and domestic investment must equal the difference between exports and imports. If the border adjustment does not affect saving and investment (more on that below), it cannot change the difference between exports and imports. If the quantities do not change, the price (the value of the dollar) must adjust to keep the equilibrium. (Other features of the reform package could change saving and investment.) If the theory is correct and the exchange rate fully adjusts – i.e., rises by the level of the tax – the border adjustment would have no effect on the trade balance, the level of exports, the level of imports, the domestic price level, or the net profitability of importers and exporters. (Again, other parts of the reform proposal could affect these items, though.) The rise in the dollar’s value would create a substantial transfer of wealth from Americans holding assets overseas (for example, in their retirement accounts) to foreigners holding assets in the United States. It could also mean trouble for emerging-market debtor countries that hold debt in dollar-denominated terms, but hold their assets in their own currencies. This is similar to what happened in the Asian financial crisis in the late 1990s. There are numerous skeptics, however, both inside and outside the economics profession on whether exchange rates would adjust fully. They highlight two concerns with the “simple economic theory” – the “simple” and the “theory” parts. The first simple example given above assumes that the border adjustment would not affect capital market flows. But it probably would, at the very least, because international investors would want to rebalance their portfolios, once their dollar-denominated investments rose by 25 percent. The second example assumes that the border adjustment would not affect saving and investment. But it probably would since it would raise revenue for the government (because imports are greater than exports) over the next 10 years and so would affect government saving. In addition, a number of factors make it difficult to estimate the exchange rate effects of border adjustment. Exchange rates can be excessively volatile, especially if the prices of goods and services adjust slowly to new events. The amount of foreign exchange related to trade flows is dwarfed by the amount related to asset purchases and sales. Some governments aim to control their exchange rates. (Although any country that does not let the dollar rise will just be making itself less competitive.) Pre-existing contracts may muddy exchange rate dynamics. Investors may hedge their bets until WTO resolution occurs. Plus, the fact that U.S. exports and imports are almost exclusively invoiced in dollar terms tends to dampen price responses to exchange rates. In any case, achieving complete exchange rate adjustment is dependent on all business tax rates being equal. If there are different rates for corporations (20 percent) and pass-through entities (25 percent), as under the House plan, then it is impossible to have a single “full” offsetting exchange rate change. If the exchange rate does not adjust fully and immediately, though, the effects of the border adjustment could be quite different. Exports would rise, imports would fall, and the trade deficit would fall. Consumer prices would rise, fueled by higher import costs, and this would hit low-income households disproportionately. Exporters would enjoy larger profits, while importers – most notably, in apparel, oil, and retailing – would be hurt. The short-term revenue yield of the tax would decline (since the tax on imports would bring in less and the subsidy for exports would be more expensive). There would be less wealth transfer from Americans to foreigners, and less new debt troubles for emerging-market countries. What would actually happen to exchange rates? Convincing empirical evidence is difficult to come by. The analysis is tricky, because financial markets can anticipate legislative changes, so that much or most of any adjustment may well have occurred before a border adjustment takes effect. There is suggestive evidence of full exchange rate offset for border-adjustable taxes but it comes from data from smaller countries who initiated smaller border-adjustable taxes than the U.S. is considering. My sense – rather than my firm conviction – given the theory and evidence to date, is that it is likely that border adjustment will create rapid and significant – maybe even close to full – exchange rate adjustments. But there is still a nontrivial risk that it would not, and that is what is keeping opponents awake at night. It would amount to a tax on imports, and in turn on the American family. Sen. Tom Cotton, R-Ark., in Washington on Jan. 10, 2017. (Photo: Chris Kleponis, AFP/Getty Images) The U.S. tax code is a mess, and Congress should eliminate all the special-interest loopholes in the code and use that money to cut taxes for everybody — including American businesses. Right now, some of my colleagues are working on a tax reform bill that would do just that. But I have grave reservations about one idea that’s being considered. It’s called a border adjustment tax, and here’s how it would work: We’d cut the corporate tax rate, and to make up for the lost revenue, we’d tax businesses whenever they bought something from another country. So, for instance, every time Ford bought an auto part from Canada, it would pay a 20% tax. Or every time your local grocery store brought in bananas from Guatemala, it would pay a 20% tax. And whatever money businesses made from selling their products in other countries would be exempt. But what all of this would really amount to is a 20% tax on imports. The proponents of this tax contend it will stop businesses from leaving our country — because right now, some are moving overseas to avoid paying our corporate tax rate, which is the highest in the modern, industrial world. Under this proposal, it wouldn’t matter where you put your headquarters. You’d be taxed according to what you bought, not where you put down stakes. The hope is this arrangement would mean more headquarters and more factories — and the jobs that come with them — would stay in America. Which of course is a desirable goal. But I don’t think this is the best way to do it. Consider this: it’s estimated that this one change alone would produce an estimated $100 billion a year in additional revenue. That’s a lot of money, and someone has to pay it. That someone is going to be working Arkansans and Americans who’ve been struggling for decades. A tax on imports is a tax on things working people buy every day — and I’m not talking about caviar and champagne. I’m talking about T-shirts, jeans, shoes, baby clothes, toys, groceries. I’ve heard from thousands of Arkansans who are already struggling just to get by. Why would we make the goods they buy at Walmart more expensive? Its supporters say this tax won’t increase the cost of imports. They argue exports will be cheaper because we’ll no longer be taxing them. They say more people overseas will buy them, meaning they’ll need more dollars with which to purchase them, which will ultimately raise the value of the dollar. According to their logic, imports won’t be more expensive because you’ll be able to buy more of them with a stronger dollar. Even with the new tax added on, you’ll still come out right where you were before. POLICING THE USA: A look at race, justice, media But this is a theory wrapped in a speculation inside a guess. Nobody knows for sure what will happen because currency markets fluctuate daily based on millions of decisions and events. Just because an economist slaps an equation on a blackboard doesn’t make it real. I’m concerned that these predictions simply won’t pan out. If we increase the costs of goods, people obviously won’t be able to buy as much, which will hurt retail sales — and retail jobs too. The retail industry is the largest employer in the U.S., and its impact is broad. In Arkansas, for instance, retail supports one in every five jobs. Are we really going to impose a huge tax on the livelihoods of so many and say, “Oh, don’t worry; it’ll all work out in the end”? That’s not a gamble many Arkansans I know would take. That’s why we have to take a hard look at this proposal now. I support fundamental tax reform and will reserve judgment on any final bill until I read it. But we must take a long, hard look at any plan that includes a border-adjustment tax. A bill that raises working families’ taxes and increases the price of the things they buy every day is hardly the way to create opportunity for all Americans. Sen. Tom Cotton, R-Ark., is a member of the Senate Armed Services Committee. You can read diverse opinions from our Board of Contributors and other writers on the Opinion front page, on Twitter @USATOpinion and in our daily Opinion newsletter. To respond to a column, submit a comment to letters@usatoday.com. Read or Share this story: http://usat.ly/2nwMBQY President-elect Donald Trump criticized a cornerstone of House Republicans’ corporate-tax plan, which they had pitched as an alternative to his proposed import tariffs, creating another point of contention between the incoming president and congressional allies. The measure, known as border adjustment, would tax imports and exempt exports as part of a broader plan to encourage companies to locate jobs and production in the U.S. But Mr. Trump, in his first comments on the subject, called it “too complicated.”... House Republicans are proposing a major reform called a border adjustment tax. President Trump was against it, but he's warming up to it. It could come up in his address to Congress on Tuesday night. And if it passes, it'll be a big deal for American consumers. It's complicated, but stick with us. Here's what you need to know. What is it? A border adjustment tax would give tax breaks to American companies that ship products to other countries, and it would strip away tax breaks from American companies that import goods from other countries. What is it supposed to accomplish? As President Trump would say: Bring the jobs back. A border adjustment tax is supposed to encourage companies to make goods in the United States. It's meant to remove the incentive for companies to move jobs overseas solely for tax reasons. Initially, a border adjustment tax makes it cheaper to export goods and more expensive to import products from abroad. Related: Trump vs. Trump on the global economy Here's the catch The U.S. dollar has to go up -- bigly. The whole thing only works if the dollar rises roughly 20% in value, and quickly. That's a big bet. A 1% move in the dollar on any day is a major swing. The thinking is that a border adjustment tax would create such an incentive for companies to make things in America that it would drive up demand for American-made goods. Higher demand would push up the value of the dollar. If the dollar rises 20%, then the benefits for companies -- whether they import or export -- basically go back to where they started. But the incentive to move jobs abroad for tax reasons is still gone. Of course, if the dollar doesn't rise 20%, prices for consumers will go up, according to one analysis by the New York Federal Reserve. The National Retail Federation, a staunch opponent, predicts everyday items you buy will get a lot more expensive. More on that below. Related: NY Federal Reserve economists criticize border adjustment tax What do supporters say? The dollar will rise 20% in value and companies will be encouraged to produce in the United States. With a stronger dollar, American companies that import will have more buying power and Main Street Americans won't see prices go up. For companies that export, it will be cheaper for them to assemble and manufacture goods. What does Trump say? He seems to be getting closer to supporting a border adjustment tax, after trashing it right before he took office. On Jan. 16, he told The Wall Street Journal, "Anytime I hear border adjustment, I don't love it." But last week he told Reuters, "It could lead to a lot more jobs in the United States." Trump has not given the idea a full endorsement. He has floated the idea of a 20% tax, or tariff, on all goods coming in from Mexico. He's also threatened tariffs against China. A border adjustment tax is considered a less threatening alternative. Related: Mnuchin promises tax reform by August What do the haters say? Retailers are bugging out. Even the Federal Reserve's economists don't like it. They say prices for clothes, TVs, cell phones and other everyday items would go up significantly. The National Retail Federation forecasts that prices would rise 15%, costing the average American family an extra $1,700 a year. Many economists are skeptical about a border adjustment tax because they don't believe the dollar will rise 20%, or that it will rise fast enough. The issue is that the dollar is influenced by many things. It's sort of like golf. Let's say you have to make a hole-in-one on the 18th hole to win a championship. Sure, you have to hit the ball perfectly. But the golf ball's destination also depends on the wind, the texture of the grass, the slope of the green... you get the idea. The dollar has its own influences, not just tax policy. It's hard to predict, let alone make, a hole-in-one on tax reform. For the dollar, its wind, grass and slope are things like Fed rate hikes, commodity prices and the strength of the U.S. economy. For companies that ship abroad, there's concern that if the dollar rises 20% in value, their products would become too expensive for foreign buyers. In 2015, the U.S. manufacturing sector went into recession because of a sharp increase in the dollar. It's also unclear whether the World Trade Organization, which includes the United States, would approve a border adjustment tax. If the WTO rejects the idea but the United States goes ahead, other countries can sue the United States through the WTO. | Summary: With an ObamaCare alternative off the table for now, the White House is turning its attention to a new initiative, the first fundamental overhaul of the tax code in 30 years. Intense lobbying already is underway, with one of the most contentious issues the idea of a "border adjustment tax," or BAT. Here's where things stand: The border tax would effectively levy a tax on products coming into the US and give tax breaks to companies sending products abroad. Proponents say it would protect US jobs, while opponents say it would raise prices of everyday goods for many Americans. See a primer at CNN. As you might expect, companies such as Boeing, Merck, and Dow Chemical that export many of their products are big fans of the BAT, while heavy importers such as retailers Walmart and Target oppose it because they say it would drive up their prices, reports the Atlantic. A huge ad war is unfolding. The National Retail Federation, for example, is running ads against the BAT, like this parody of an infomercial. Paul Ryan and House Republican leaders are pushing for the BAT, but their Senate counterparts generally oppose it, reports the the Washington Post. Conservative Tom Cotton, for instance, called it "a theory wrapped in a speculation inside a guess," in this op-ed at USA Today. In a possible hint of compromise, the Post notes that Treasury chief Steven Mnuchin has suggested that some products or industries could be exempt, without offering details. President Trump himself sounded skeptical of the tax initially but might be warming up. "Anytime I hear border adjustment, I don't love it," he told the Wall Street Journal in January. In February, however, he told Reuters that it "could lead to a lot more jobs," and on Sunday, Reince Priebus said Trump thought a border tax could even "the playing field between our country" and others, per Fox News. The success of the BAT hinges on the economic premise that it would strengthen the US dollar, though there's a fair amount of skepticism about that, notes Business Insider. A more in-depth analysis, which takes note of "significant confusion and uncertainty" about how exchange rates might respond, is at Real Clear Economics. The European Union might sue if a border tax goes into effect, reports dw.com. Canada isn't a fan, either, notes Fortune. More broadly, the GOP's failure to repeal ObamaCare complicates tax reform, because Republicans were relying on savings from the ACA's elimination as part of their calculations. The New York Times delves into the thorny legislative consequences, which mean that Republicans might have to work more closely with Democrats. | 7,451 | 578 | multi_news | en |
Write a title and summarize: Deregulated CD8+ T cell cytotoxicity plays a central role in enhancing disease severity in several conditions. However, we have little understanding of the mechanisms by which immunopathology develops as a consequence of cytotoxicity. Using murine models of inflammation induced by the protozoan parasite leishmania, and data obtained from patients with cutaneous leishmaniasis, we uncovered a previously unrecognized role for NLRP3 inflammasome activation and IL-1β release as a detrimental consequence of CD8+ T cell-mediated cytotoxicity, ultimately resulting in chronic inflammation. Critically, pharmacological blockade of NLRP3 or IL-1β significantly ameliorated the CD8+ T cell-driven immunopathology in leishmania-infected mice. Confirming the relevance of these findings to human leishmaniasis, blockade of the NLRP3 inflammasome in skin biopsies from leishmania-infected patients prevented IL-1β release. Thus, these studies link CD8+ T cell cytotoxicity with inflammasome activation and reveal novel avenues of treatment for cutaneous leishmaniasis, as well as other of diseases where CD8+ T cell-mediated cytotoxicity induces pathology. Granule mediated cytotoxicity is required for the clearance of several viral pathogens, as well as the killing of tumor cells [1]. However, cytotoxicity can also provoke a detrimental inflammatory response in several diseases, including experimental cerebral malaria, Trypanosoma cruzi-elicited cardiomyopathy and Coxsackievirus B3-induced myocarditis [2–6], and can contribute to the pathology of atherosclerotic disease, rheumatoid arthritis, chronic kidney disease, diabetes and atopic dermatitis [7–16], as well as many forms of drug-induced cutaneous hypersensitivity [17]. While we have a good understanding of the mechanism by which cytotoxicity leads to viral clearance and the control of malignant transformed host cells, how CD8+ T cell-mediated killing of infected cells leads to tissue inflammation is still poorly understood. Cutaneous leishmaniasis, caused by an intracellular protozoan parasite transmitted by sand flies, exhibits a wide spectrum of clinical manifestations. There is no vaccine for leishmaniasis, and chemotherapeutic drugs are toxic and often ineffective. Some of the most severe forms of the disease occur in Brazil, where patients develop chronic single or multiple ulcerated lesions, and in some cases a disfiguring form of the disease called mucosal leishmaniasis. Somewhat surprisingly, these severe forms of leishmaniasis are not driven by a high parasite burden, but rather are due to an uncontrolled inflammatory response [18]. For a long time it was believed that the disease was driven by a poorly regulated CD4+ Th1 response, leading to exaggerated inflammation. However, recent findings demonstrate that the inflammation seen in L. braziliensis patients is strongly associated with granule-mediated cytotoxicity induced by CD8+ T cells [19–25], and recent studies in mice conclusively demonstrated that CD8+ T cell-mediated cytotoxicity is a cause rather than a consequence of pathology in cutaneous leishmaniasis [23] [26,27]. These findings suggest that targeting CD8+ T cell cytotoxicity for an immunotherapy might be protective, an approach far better than blocking a CD4+ Th1 response that could lead to uncontrolled parasite replication. However, to develop such a therapeutic approach requires defining the pathway that leads to severe pathology by cytolytic CD8+ T cells. CD8+ T cell-induced apoptosis of target cells is generally not considered inflammatory, since the intracellular content of the dying cells is confined to apoptotic bodies that are rapidly engulfed by neighboring phagocytes [28]. However, there is increasing evidence that apoptosis is not always ‘silent’ and can also be immunogenic [28]. Specifically, release of “danger signals” from dying cells can activate inflammasomes, multiprotein complex sensors that regulate the processing of caspase-1 to activate pro-inflammatory cytokines such as IL-1β [29]. In support, a genome-wide transcriptional profiling of lesions from L. braziliensis patients compared to normal skin revealed that genes involved in both cytotoxicity and inflammasome activation were highly upregulated[24]. Furthermore, both inflammasome activation and IL-1β have been linked with disease severity in leishmaniasis [30], suggesting that CD8+ T cell cytotoxicity might increase inflammasome activation and IL-1β production, thereby driving disease severity. Here we show that inflammasome activation and IL-1β release is indeed driven by CD8+ T cell-induced cytotoxicity. By employing two different murine models of infection, we found that CD8+ T cell-induced pathology depended on the NLRP3 inflammasome and IL-1β signaling, and demonstrated that the NLRP3 inflammasome is required for the high levels of IL-1β present within lesions of leishmaniasis patients. Furthermore, we demonstrated that CD8+ T cell-induced pathology could be abrogated with pharmacological inhibitors of NLRP3 or IL-1, which opens up the possibility of using several FDA-approved, commercially available drugs to ameliorate disease in patients. Together, these results provide the foundation for new strategies for treating leishmaniasis patients, as well as other diseases where CD8+ T cell-cytotoxicity drives pathology. In order to define the downstream mechanisms of CD8+ T cell cytotoxicity that cause immunopathology following infection with L. braziliensis we utilized our recently developed murine model [23]. As we previously reported, RAG deficient mice infected with L. braziliensis do not develop lesions in the skin despite being unable to control parasites (Fig 1A) [23]. In contrast, while RAG deficient mice reconstituted with CD8+ T cells (RAG+CD8) and infected with L. braziliensis remain unable to control the parasites [23], they now develop severe lesions over the course of weeks (Fig 1A). In addition to containing CD8+ T cells, the lesions of RAG+CD8 mice had more CD11b+ cells than control RAG mice at 7 weeks (mean number of CD11b+ cells—naïve RAG: 2. 4 x 104; infected RAG: 5. 3 x 104; RAG+CD8: 32 x 104). Notably, both Il1a and Il1b RNA expression were significantly increased in RAG+CD8 mice in comparison to RAG mice, though the increase in Il1b was much greater than Il1a (Fig 1B). We next asked if pro-IL-1β protein was also expressed in the skin by flow cytometry. Infection of RAG mice with L. braziliensis did not change the expression of pro-IL-1β in the skin, suggesting that parasites alone do not induce pro-IL-1β expression at the infection site (Fig 1C and 1D). Conversely, RAG+CD8 mice had a significant increase in the frequency of CD11b+ cells expressing pro-IL-1β (Fig 1C and 1D) and there was enhanced secretion of IL-1β from ears of RAG+CD8 mice after 48 hours of in vitro culture as measured by ELISA (Fig 1E). With the exception of dendritic cells, all populations of myeloid cells analyzed, including macrophages, monocytes and neutrophils expressed more pro-IL-1β after infection in RAG+CD8 mice compared with RAG mice (S1A–S1D Fig). Notably, the pathology induced by CD8+ T cells was associated with increased recruitment of neutrophils to the skin, and the frequency of Ly6G+ cells was significantly higher in RAG+CD8 mice compared to RAG mice (S1E and S1F Fig). Therefore, the majority of cells expressing pro-IL-1β were monocytes in RAG mice, whereas in RAG+CD8 mice, neutrophils accounted for more than 70% of the IL-1β production within the skin (S1G Fig). The increased inflammation observed in RAG+CD8 mice was associated with a significant increase in mRNA levels for CCL3 and CXCL1 (S2 Fig) suggesting that CCL3 and CXCL1 might be responsible for the intense recruitment of neutrophils induced by CD8+ T cells. The pathology induced by CD8+ T cells is dependent on granule-mediated cytotoxicity, since L. braziliensis-infected RAG mice reconstituted with perforin deficient CD8+ T cells (RAG+PRF-/-CD8) do not develop pathology (Fig 1F) [23]. Therefore, we next asked if pro-IL-1β was decreased in cells from infected RAG+PRF-/-CD8 mice. In fact, pro-IL-1β expression after L. braziliensis infection in RAG+PRF-/-CD8, though slightly higher than RAG mice infected without T cells, was significantly lower than in RAG+WT CD8 (Fig 1G and 1H), suggesting that the increased expression of pro-IL-1β in the skin is dependent on the cytolytic activity of CD8+ T cells. To determine if cytotoxicity is also necessary for pro-IL-1β expression in other skin models of inflammation, we used an imiquimod treatment model that mimics certain aspects of psoriatic lesions in which both cytotoxicity and IL-1 have been implicated in disease [31,32]. We found that imiquimod treatment (S3 Fig) increased the frequency of pro-IL-1β-producing CD11b+ cells in WT mice but not in perforin deficient mice (S3B Fig). Together, these results demonstrate that the presence of cytolytic CD8+ T cells promotes IL-1β production, which is associated with enhanced recruitment of neutrophils and other cells to the skin, many of which also express pro-IL-1β. To test whether IL-1β was responsible for increased disease severity, or was a consequence of increased inflammatory signaling, we monitored the extent and kinetics of lesion development in RAG+CD8 mice that were treated with anti-IL-1R, anti-IL-1β or anti-IL-1α monoclonal antibodies two weeks after infection. Notably, both anti-IL-1R and anti-IL-1β treated mice developed much smaller lesions in comparison to control mice (Fig 2A and 2B). Since IL-1α is highly expressed in the skin of mice and humans, it seemed likely that it might also contribute to the development of pathology. Indeed, recent studies show that IL-1α activated by commensal bacteria in the skin amplifies the inflammatory response in leishmaniasis [33]. However, anti-IL-1α had minimal impact on the development of pathology (Fig 2C). Importantly, blockade of IL-1α, IL-1β or both (anti-IL-1R) had no effect on CD8+ T cell production of GzmB or IFN-γ (Fig 2D), or on parasite numbers (Fig 2E). To determine if a well-established treatment for patients with IL-1 dependent inflammatory diseases might be an effective immunotherapy in cutaneous leishmaniasis, we treated RAG+CD8 mice with anakinra, a recombinant version of the IL-1R antagonist [34]. Critically, pharmacological blockade of IL-1 signaling prevented the severe CD8+ T cell-mediated pathology normally present in RAG+CD8 mice (Fig 2F), and again had no impact on GzmB or IFN-γ expression by CD8+ T cells (Fig 2G) or parasite numbers (Fig 2H). In addition, we also found that treatment with anti-IL-1R mAb decreased the lesions size of BALB/c mice infected with L. braziliensis without affecting parasite numbers (S4A and S4B Fig). Together, our data reveal that pathology induced by CD8+ T cells in the skin is dependent on IL-1β. Since blockade of this cytokine does not affect IFN-γ production or parasite control, our studies identify anakinra or monoclonal antibodies that specifically block IL-1β (such as canakinumab) as potential therapeutics for treatment of cutaneous leishmaniasis. Furthermore, since GzmB levels were not altered by IL-1 blockade, our results suggest that IL-1β production occurs downstream of CD8+ T cell activation. In order to ask if blockade of IL-1 was effective after the onset of pathology, we started the treatment with anakinra after signs of inflammation, such as redness and thickening of the skin, had started to develop in RAG+CD8 mice. We found that treatment after pathology has already developed in RAG+CD8 mice results in slower lesion development, although it does not completely prevent inflammation (S4C Fig). Treatment after the onset of pathology did not affect parasite control (S4D Fig). The CD8+ T cell-mediated pathology seen in human leishmaniasis is not recapitulated in most murine models of leishmania infection. However, we recently found that co-infection with acute lymphocytic choriomeningitis virus (LCMV) in C57BL/6 mice leads to exacerbated skin immunopathology in L. braziliensis-infected mice, which depends on CD8+ T cells, and not NK cells or CD4 T cells [26]. In this model, L. major infected mice are infected systemically with LCMV two weeks post-infection. In spite of a co-infection, as in singly infected mice LCMV clearance occurs by day 8, and there is only a transient increase in the leishmania burden of co-infected animals [26]. However by 5 weeks post L. major infection, co-infected mice develop larger leishmanial lesions than do controls, with a significant increase in the frequency of neutrophils. This model, initially developed to demonstrate that viral co-infections can alter the magnitude of disease in leishmaniasis patients, allows us to probe the mechanisms of pathology mediated by CD8+ T cells in conventional animals with a full complement of immune cells. Therefore, we first determined if IL-1 was also required in this model of CD8+ T cell-dependent pathology, and treated co-infected and control mice with anakinra starting 24 days after L. major infection. As previously reported [35], the course of infection with L. major is not altered in the absence of IL-1 (Fig 2I). Importantly, however, treatment with anakinra 10 days post LCMV infection completely abrogated leishmanial-induced skin pathology (Fig 2I), despite similar parasite burdens, frequency of IFN-γ-producing CD4+ and CD8+ T (Fig 2G, 2K and 2I), and GzmB-expressing CD8+ T cells (Fig 2G and 2I). Altogether, these data demonstrate that IL-1 signaling plays an unexpected and key role in severe disease pathology in murine models of leishmaniasis driven by cytotoxic CD8+ T cells. IL-1β requires processing to become active and signal through the IL-1R [36]. There are both inflammasome dependent and independent pathways to process IL-1 and specifically, neutrophil proteases have been demonstrated to be sufficient to cleave IL-1β in situations where these cells are abundant [37] [38] [39], which is the case in L. braziliensis lesions. Therefore, to determine if the inflammasome was involved in disease caused by CD8+ T cells, we infected WT or caspase-1/11 deficient C57BL/6 mice with L. major. As previously reported [40], caspase-1/11 deficient mice infected with L. major had similar lesion development as WT mice (Fig 3A). In contrast, while WT mice developed larger lesions after co-infection with LCMV, lesions in caspase-1/11 deficient mice were smaller at 4 weeks post infection (Fig 3A). Though it is not clear if those responses are leishmania-specific, deficiency in caspases-1/11 did not affect either GzmB or IFN-γ production by CD8 or CD4 T cells (Fig 3B) and parasite numbers detected at 5 weeks post-infection remained unchanged (Fig 3C). Together, these data suggest that caspase-1/11 is required for pathology induced by CD8+ T cells and importantly does not affect parasite control. Under certain conditions caspase-11 can contribute to IL-1β processing, but regardless of the stimulus caspase-1 appears to be required [41]; at present we do not know if caspase-11 also contributes to IL-1β release in this model. Since caspase-1 plays a critical role, and can be cleaved by several different inflammasomes in order to become active [36], we next investigated which inflammasome is required for CD8+ T cell-mediated pathology. We focused on the NLRP3 inflammasome, as NLRP3 mRNA levels were significantly higher in lesions from L. braziliensis patients in comparison to normal skin [24], and NLRP3 can be activated by damage-associated molecular patterns (DAMPs) [42], potentially released by CD8+ T cell-induced cell death. We found that following infection of WT or NLRP3 deficient C57BL/6 mice with L. major, WT and NLRP3 deficient mice had similar lesion sizes (Fig 3D). However, while WT mice co-infected with LCMV developed severe lesions, NLRP3 deficiency decreased CD8+ T cell-mediated disease (Fig 3D). The changes in pathology were not associated with differences in the capacity of either CD4 or CD8+ T cells to make IFN-γ, or CD8+ T cells to express GzmB (Fig 3E). Importantly, the number of parasites in the skin was similar in all groups of mice (Fig 3F). These differences were not associated with a deficit in the ability of CD8+ T cells from NLRP3 deficient mice to respond since CD8+ T cells from NLRP3 deficient mice infected with LCMV alone responded as well as CD8+ T cells from wild-type mice (S5A–S5D Fig). The NLRP3 inflammasome has been previously linked with expression of inducible nitric oxide synthase (iNOS) [40], here we found no significant changes in CD11b+ cells expressing iNOS by flow cytometry (S6 Fig). Together, these results suggest that IL-1β release is dependent upon the NLRP3 inflammasome, which is particularly important as the NLRP3 inflammasome has been linked to chronic inflammation [43] and new drugs are being developed to block its activation. Our results above suggest that the NLRP3 inflammasome is activated by CD8+ T cell-mediated cytotoxicity and drives disease progression. A number of small molecule inhibitors have been identified that inhibit NLRP3 inflammasome activation, for example, MCC950 and the diabetes drug glyburide [44] [45]. We therefore asked if blocking this pathway after leishmania infection could influence the progression of disease. Indeed, co-infected mice treated with MCC950 or glyburide failed to develop the severe disease seen in untreated co-infected mice (Fig 4A and 4B), while treatment with NLRP3 inhibitors had no effect on mice only infected with leishmania (Fig 4A and 4B). Critically, smaller lesions in mice treated with the NLRP3 inhibitors was associated with a decrease in neutrophils present in the skin in comparison to control mice co-infected with LCMV (Fig 4C and 4D). Inhibiting the NLRP3 inflammasome in co-infected mice had no significant effect on parasite numbers (Fig 4E and 4F), suggesting once again that reduction in disease severity was unrelated to parasite control. To confirm these results, we treated RAG+CD8 mice with glyburide or MCC950 2 weeks post L. braziliensis infection. Similar to the co-infected mice, pharmacological blockade of the NLRP3 inflammasome significantly reduced lesion development in RAG+CD8 mice (S7A Fig) without affecting parasite control in the skin (S7B Fig). Using a genome-wide transcriptional profiling in L. braziliensis patients [24], we found that both IL1A and IL1B transcripts were elevated compared with normal skin (S8A and S8E Fig). Importantly, we found that levels of IL1B expression were positively correlated with those of GZMB, GZMA and PRF1 (S8B, S8C and S8D Fig), whereas IL1A expression did not correlate with GZMB, GZMA or PRF1. The high levels of IL-1β mRNA correlated with increased expression of pro-IL-1β in the skin (Fig 5A). Similar to what we observed in mice, expression of pro-IL1β is different between granulocytes (CD11b+ CD66b+ cells) and macrophages/monocytes (CD11b+ CD68+ cells) in the skin of L. braziliensis patients (Fig 5B). To determine if IL-1β was processed and released by cells in leishmanial lesions, we collected lesion biopsies from L. braziliensis patients, cultured them in vitro for 48 hr, and measured the amount of IL-1β protein released into the supernatant. We found elevated levels of IL-1β in the supernatants of cultured L. braziliensis skin biopsies but not normal skin biopsies (Fig 5C). Finally, to determine if IL-1β production in human lesions was dependent on the NLRP3 inflammasome, we tested if IL-1β production by skin biopsies from L. braziliensis lesions was blocked by NLRP3 inhibition. Biopsies were again collected from patients, and then divided into two, with one half acting as a control, and the other treated with glyburide. While untreated skin lesion biopsies produced IL-1β, treatment with glyburide significantly decreased the release of IL-1β in culture (Fig 5D). Importantly, treatment with glyburide did not affect the production of other cytokines, such as IL-6 and IL-10 (Fig 5E and 5F). Together, these findings show evidence that the pathway identified in mice leading from CD8+ T cell cytotoxicity to NLRP3 inflammasome activation and subsequently IL-1β release may also be occurring in L. braziliensis patients. Thus, our results strongly suggest that targeting the inflammasome or IL-1β in humans with existing therapeutic regimens could prevent the pathology induced by excessive CD8+ T cell-mediated cytotoxicity. The severity of disease progression following an infection depends not only on the pathogen burden, but also on the magnitude and type of the inflammatory response elicited. This is especially true for certain forms of cutaneous leishmaniasis, where disease progression can be relatively independent of the number of parasites and is instead driven by an exaggerated immune response. Though it is clear that cytotoxic CD8+ T cells causes immunopathology in cutaneous leishmaniasis [19] [20,21,25] [24] [23,26,27], a fundamental understanding of how cytotoxicity enhances inflammation and disease progression was undetermined. In this study, we have dissected the downstream mechanisms of CD8+ T cell cytotoxicity that exacerbates the inflammatory skin environment and found a central role for the NLRP3 inflammasome and IL-1β. Besides increasing our understanding of how cytotoxicity influences inflammation, this work provides evidence for previously unappreciated targets to suppress immunopathology in cutaneous leishmaniasis and other situations in which cytotoxicity exacerbates disease. IL-1 has been linked to disease in many chronic inflammatory disorders and while IL-1 can have protective effects, the preponderance of data in leishmania infection indicates that excessive IL-1 is detrimental. Thus, while early production of IL-1 enhances T cell priming and promotes the development of protective Th1 responses during leishmania infection [46] [47] [48], continuous treatment of infected mice with IL-1 exacerbates disease [48] [47], and a recent study with a non-healing strain of L. major found that IL-1β was required for the chronic disease phenotype [30]. Furthermore, increased disease severity in patients infected with L. mexicana has been linked with the excess IL-1β production [49]. Taken together, the combined results of several studies indicate that while healing can occur in the absence of IL-1, the overproduction of IL-1β promotes disease severity. The canonical pathway for maturation and release of IL-1β involves processing of pro-IL-1β by caspase-1, following activation of caspase-1 by the inflammasome. Since the NLRP3 inflammasome can be activated by DAMPs released from dying cells, and the NLRP3 gene was upregulated in lesions from L. braziliensis patients, [24] we hypothesized that CD8-T cell mediated cytolysis drives disease progression by increasing NLRP3 inflammasome-driven caspase-1 activation. Indeed, our results show that pathology was reduced in caspase-1/11 and NLRP3 knockout mice. These results are consistent with two previous studies showing that NLRP3 inhibits protective immune responses in leishmaniasis [30,50]. In contrast, one study has reported that NLRP3 deficient mice infected with L. amazonensis are more susceptible to infection, although L. amazonensis infected mice fail to heal in the presence or absence of the NLRP3 inflammasome [40]. Since mice infected with L. amazonensis develop a poor CD4+ Th1 response [51], it may be that under these circumstances NLRP3-dependent IL-1β promotes better protection by activating the iNOS pathway of parasite control [40]. Here we found no evidence that iNOS expression is different between NLRP3 sufficient and deficient mice, suggesting that at least in CD8+ T cell-induced pathogenesis NLRP3 deficiency does limit iNOS expression in the skin. In addition to finding that NLRP3 knockout mice developed less severe disease, we also found that inhibitors of the NLRP3 inflammasome protected mice from developing the larger lesions caused by cytotoxic CD8+ T cells. MCC950 is a small molecule that selectively inhibits the NLRP3 inflammasome and attenuates disease severity in a number of experimental autoinflammatory disease models, including murine experimental autoimmune encephalomyelitis and cryopyrin-associated autoinflammatory syndrome [44]. Glyburide, a drug commonly used in type 2 diabetes, can also prevent NLRP3-dependent IL-1β production [45]. Intriguingly, both MCC950 and glyburide prevented the development of severe lesions in leishmania-infected mice. Importantly, both genetic and pharmacological inhibition with NLRP3 inflammasome activation blocked immunopathology while keeping immunoprotective responses intact as observed by similar production of IFN-γ by T cells and equivalent parasite numbers in the skin. Particularly important for translation of our findings in mice to patients, glyburide also inhibited production of IL-1β in infected human lesion biopsies, indicating that production of IL-1β in human leishmania lesions is also dependent on NLRP3. Together, these results demonstrate that pharmacological blockade of the NLRP3 inflammasome dampens the immunopathology caused by cytotoxic CD8+ T cells, and may be an effective strategy to ameliorate disease in L. braziliensis patients. The role of neutrophils in cutaneous leishmaniasis is complex, and depends upon particular host-parasite combinations, as well as the stage of the infection [52]. Thus, they can promote increased protection, but in other circumstances promote increased susceptibility [53–55], [56,57]. However, in chronic leishmaniasis it appears that neutrophils are primarily associated with increased disease [23,26,27,30], which occurs in the severe leishmanial lesions that develop due to excessive CD8+ T cell cytolysis. Persistent recruitment of neutrophils, which in our mouse model is associated with increased expression of CCL3 and CXCL1, drives pathology in other types of infections as well [58], by releasing matrix metalloproteases, reactive oxygen species, and myeloperoxidase all of which are significantly increased in L. braziliensis human biopsies in comparison to normal skin [24]. Notably, neutrophil-released effector molecules amplify the response of neutrophils and increase neutrophil recruitment to sites of inflammation, thereby promoting tissue damage [59]. In addition, cytokines, pathogen-associated molecular patterns and DAMPs present at inflammatory sites prolong the life span of neutrophils, contributing to chronic inflammation [60] [61] [62]. In addition to directly promoting disease, here we found that neutrophils are a major source of IL-1β during chronic infection. Thus, the recruitment of neutrophils may lead to a feed-forward loop that amplifies and sustains the inflammation in the lesions of cutaneous leishmaniasis patients. CD8+ T cell cytotoxicity is damaging in many conditions, including drug-induced cutaneous hypersensitivity [17], experimental cerebral malaria, Trypanosoma cruzi-elicited cardiomyopathy and Coxsackievirus B3-induced myocarditis [2–6], and contributes to the development of artherosclerotic disease, rheumatoid arthritis, chronic kidney disease, diabetes and atopic dermatitis [7–16]. Whether the inflammation mediated by CD8+ T cell cytotoxicity is dependent on inflammasome activation in those conditions remains to be determined, although IL-1β has been implicated in driving disease severity in some of these instances [32,63–66]. Particularly relevant to our findings, IL1b mRNA levels are decreased in perforin or granzyme B deficient mice compared to WT mice in a mouse model of atherosclerosis [67]. Interestingly, cytotoxicity is enhanced in psoriatic lesions of human patients [31,68] and patients with psoriasis have been successfully treated with anakinra [69,70], further suggesting that cytotoxicity may drive IL-1-induced inflammation in this condition. Taken together, these findings suggest that the mechanism of inflammation induced by cytotoxic CD8+ T cells is not unique to L. braziliensis infection and may contribute to immunopathology in several other diseases. Therapy for cutaneous leishmaniasis can be toxic and is often ineffective. This may in part be due to the fact that the drugs target the parasites but not the immunopathologic responses mediating some of the most severe forms of the disease. Using a combination of murine models and studies in cutaneous leishmaniasis patients, we have identified the NLRP3 inflammasome and IL-1β as components of the pathologic response that occurs as a consequence of CD8+ T cell cytotoxicity in severe forms of leishmaniasis. Importantly, these immune responses do not have an effect on parasite numbers. Furthermore, patients treated early during L. braziliensis infection are surprisingly less responsive to drug treatment when compared with patients that have already developed more severe lesions [71]. These patients also already have evidence of increased IL-1β [24]. Therefore, blocking IL-1β might be a valuable strategy to treat patients at the early onset of disease. Thus, blocking IL-1β with drugs currently being used clinically for other chronic diseases, such as anakinra and canakinumab, or inhibiting NLRP3 inflammasome activation, in combination with anti-parasitic drugs would ameliorate disease severity while sparing immunoprotective responses in cutaneous leishmaniasis. In summary, this work reveals a previously unrecognized link between CD8+ T cell mediated cytotoxicity and the well-known pathway of IL-1β mediated inflammation, which has important implications for patients with severe forms of cutaneous leishmaniasis. This study was conducted according to the principles specified in the Declaration of Helsinki and under local ethical guidelines (Ethical Committee of the Maternidade Climerio de Oliveira, Salvador, Bahia, Brazil; and the University of Pennsylvania Institutional Review Board). This study was approved by the Ethical Committee of the Federal University of Bahia (Salvador, Bahia, Brazil) (010/10) and the University of Pennsylvania IRB (Philadelphia, Pa) (812026; 823847). All patients provided written informed consent for the collection of samples and subsequent analysis. This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Institutional Animal Care and Use Committee, University of Pennsylvania Animal Welfare Assurance Number 803457. All cutaneous leishmaniasis patients were seen at the health post in Corte de Pedra, Bahia, Brazil, which is a well-known area of L. braziliensis transmission. The criteria for diagnosis were a clinical picture characteristic of cutaneous leishmaniasis in conjunction with parasite isolation or a positive delayed-type hypersensitivity response to leishmania antigen, plus histological features of cutaneous leishmaniasis. Prior to therapy, biopsies were collected at the border of the lesions using a 4 mm punch before therapy. Biopsies were treated with collagenase for 90 mins at 37°C/5% CO2, dissociated and passed through a 50 μm Medicon filter (BD phamingen). Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood layered over a Ficoll-Hypaque gradient (GE Healthcare), then washed by centrifugation and resuspended in RPMI media. Biopsies were either cultured for assessment of IL-1β, IL-6 and IL-10 production or used directly ex vivo for flow cytometry. Biopsies from normal skin or leishmania lesions were not processed and the entire punch biopsy was cultured for 48 hours at 37°C/5% CO2 in RPMI supplemented with 10% human AB serum, 2 mM glutamine, 200 U/ml penicillin, and 200 μg/ml streptomycin. Where specified, the biopsies were cut into two pieces and half of the biopsies were cultured in media containing vehicle (DMSO) or 200μM of glyburide (SIGMA). Cytokine levels in the supernatants were then measured in ELISA assays (R&D Systems) according to the manufacturer’s instructions. For flow cytometry analysis, cells were stained immediately after dissociation. BALB/c and C57BL/6 mice (6 weeks old) were purchased from Charles River, and RAG-/- (B6. 12957-RAG1tm1Mom) and perforin-/- (C57BL/6-Prf1tm1Sdz) were purchased from The Jackson Laboratory. C57BL/6 Caspase-1/11-/- mice [72] and the NLRP3-/- mice [73] were provided by Dr. Richard Flavell (Yale University). All mice were maintained in a specific pathogen-free environment at the University of Pennsylvania Animal Care Facilities. L. braziliensis parasites (strain MHOM/BR/01/BA788) and L. major parasites (Friedlin) were grown in Schneider’s insect medium (GIBCO) supplemented with 20% heat-inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. Metacyclic enriched promastigotes were used for infection [74]. Mice were infected with either 105 L. braziliensis or 2x106 L. major in the left ear, and the course of lesion progression was monitored weekly by measuring the diameter of ear induration with digital calipers (Fisher Scientific). For LCMV infections, mice were infected with 2×105 PFU of LCMV Armstrong strain by i. p. injection. Splenocytes from C57BL/6 WT or perforin-/-, mice were collected, red blood cells lysed with ACK lysing buffer (LONZA) and CD8+ T cells were purified using a magnetic bead separation kit (Miltenyi Biotec). Three million CD8+ T cells were transferred into RAG mice that were subsequently infected with L. braziliensis. Mice reconstituted with CD8+ T cells received 4 injections of 250 μg of anti-CD4 within the first 2 weeks. Infected and uninfected ears were harvested, the dorsal and ventral layers of the ear separated, and the ears incubated in RPMI (Gibco) with 250 μg/mL of Liberase (Roche) for 90 mins at 37°C/5% CO2. Following incubation, the enzyme reaction was stopped using 1mL of RPMI media containing 10% FBS. Ears were dissociated using a cell strainer (40 μm, BD Pharmingen) and an aliquot of the cell suspension was used for parasite titration. The parasite burden in the ears was quantified as described previously [75]. Briefly, the homogenate was serially diluted (1: 10) in 96-well plates and incubated at 26°C. The number of viable parasites was calculated from the highest dilution at which parasites were observed after 7 days. Cell suspensions from mice were incubated with PMA (50 ng/mL), ionomycin (500 ng/mL) and Brefeldin A (10 μg /mL) (all from SIGMA) for IFN-γ and granzyme B intracellular staining. Cells suspensions were used directly ex vivo for pro-IL-1β intracellular staining. Before surface and intracellular staining, cells were washed and stained with live/dead fixable aqua dead cell stain kit (Molecular Probes), according to manufacturer instructions. Cell suspensions from human skin or PBMC were stained with flow cytometry antibodies directly ex vivo. All flow cytometry analysis was performed using the FlowJo Software. Naive ears and L. braziliensis infected ears from RAG mice with no cells or RAG+CD8 mice were excised and the ear sheets separated and incubated intact for 48 hours in complete RPMI. Supernatants were used for mouse IL-1β ELISA (Biolegend) according to the manufacturer’s instructions. 2–3 weeks post infection of RAG mice with L. braziliensis or 10 days post LCMV infection in C57BL/6 mice infected with L. major, treatment with either monoclonal antibodies or drugs commenced. Mice treated with anti-IL-1R, anti-IL-1α or anti-IL-1β (all from BioXcell) received 500 μg of antibody i. p. twice a week until the termination of the experiment. Mice were treated with 50mg/Kg of anakinra (Sobi) i. p. every day throughout the course of the experiment or 10mg/Kg of MCC950 (Sigma) and 5μM of glyburide (Sigma) i. p. every other day until the termination of the experiment. For imiquimod (Perrigo) treatment, the mouse flank was shaved the day before the first imiquimod application. WT or perforin-/- mice were treated with 62. 5mg of imiquimod (5%) in the flank and in the ear for 6 consecutive days. Mice were euthanized on the 7th day and ear were used for flow cytometry directly ex vivo as described above. Mouse: anti-CD45. 2 APC-AlexaFluor 750, anti-CD11b eF450, anti-CD11c FITC, anti-F4/80 PE-Cy7, anti-CD3 eFluor 450, anti-IL-1β pro-form and anti-IFN-γ PeCy7 (all from eBioscience). Anti-CD4 APC-Cy7 and Ly6C PerCP-Cy5. 5 (BD Pharmingen), anti-CD8β PerCPCy5. 5 and anti-Ly6G APC (Biolegend) and anti-granzyme B APC (Invitrogen). Human: anti-CD11b PeCy7, anti-IL-1β PE, anti-CD3 APCCy7 and anti-CD8a PeCy5. 5 (all from eBioscience). Anti-granzyme B APC (Invitrogen). The ear tissue was homogenized using a tissue homogenizer (FastPrep-24, MP Biomedical), and total RNA was extracted using the RNeasy Mini kit (QIAGEN) according to the manufacturer’s instructions. RNA was reverse transcribed using high capacity cDNA Reverse Transcription (Applied Biosystems). Real-time RT-PCR was performed on a ViiA 7 Real-Time PCR System (Applied Biosystems). Relative quantities of mRNA were determined using SYBR Green PCR Master Mix (Applied Biosystems) and by the comparative threshold cycle method, as described by the manufacturer. mRNA levels for each sample were normalized to Ribosomal protein S14 genes (RPSII) and displayed as fold induction over uninfected skin. Primers were designed using Primer Express software (version 2. 0; Applied Biosystems); Il1b, forward, 5′-TTGACGGACCCCAAAAGAT-3′, and reverse 5′-GATGTGCTGCTGCGAGATT-3′; Il1a, forward 5’-TTGGTTAAATGACCTGCAAC -3’, and reverse 5’-GAGCGCTCACGAACAGTTG-3’; Ccl3, forward, 5′- TGCCCTTGCTGTTCTTCTCT-3′, and reverse 5′- GTGGAATCTTCCGGCTGTAG-3′, Ccl2, forward, 5′- GCTTCTGGGCCTGCTGTTCA-3′, and reverse 5′- AGCTCTCCAGCCTACTCATT-3′; Ccl5, forward, 5′- GCAGCAAGTGCTCCAATCTT-3′, and reverse 5′-CAGGGAAGCGTATACAGGGT-3′; Ccl7, forward, 5′- AGGATCTCTGCCACGCTTC-3′, and reverse 5′- TTGACATAGCAGCATGTGGAT-3′; Cxcl1, forward, 5′- GCACCCAAACCGAAGTCATA-3′, and reverse 5′- CTTGGGGACACCTTTTAGCA-3′; Cxcl4, forward, 5′- CCATCTCCTCTGGGATCCAT-3′, and reverse 5′- CCATTCTTCAGGGTGGCTAT-3′. For transcriptional profiling, cRNA was generated from 10 normal skin and 25 lesion biopsy samples as described previously [24]. Data is deposited on the Gene Expression Omnibus (GEO) database for public access (GSE number GSE55664). Data are presented as mean ± standard error or individual samples. For mouse and human experiments statistical significance was determined using the two-tailed unpaired Student’s t-test, with the exception of paired t-test that was used for human experiments in which the same patient sample was compared between different treatments. Pearson correlation coefficient was used to determine correlation between LOG2 expressions of genes obtained from human skin transcriptional profiling. All statistical analysis was calculated using Prism software (GraphPad). Differences were considered significant when p ≤ 0. 05 (*), p ≤ 0. 01 (**) or p ≤ 0. 001 (***). | Title: CD8+ T cell cytotoxicity mediates pathology in the skin by inflammasome activation and IL-1β production Summary: Leishmaniasis is a neglected tropical disease endemic in 98 countries and approximately 1 million new cases occur each year. Disease caused by Leishmania braziliensis, the main causative agent of leishmaniasis in South America, leads to skin ulcers that are difficult to heal with drugs that target the parasites. This is because disease severity seen in patients infected with L. braziliensis is largely due to the immune response that develops, rather than the number of parasites in the skin. CD8+ T cells induce cell death in the lesions of L. braziliensis-infected mice, as well as in the lesions from L. braziliensis-infected patients, which promotes disease. However, the mechanism mediating CD8+ T cell dependent pathology is unknown. Here, using studies in mice and experiments with L. braziliensis patients' samples we show that increased disease severity is due to inflammasome activation, and furthermore that therapies that block either inflammasome activation or IL-1β ameliorate disease in mouse models of severe leishmaniasis. Based on these studies we propose a novel strategy of therapy for L. braziliensis infection and other diseases in which cytotoxicity plays a central role in promoting disease severity. | 10,259 | 303 | lay_plos | en |
Summarize: NICK JENSEN’s wife Sarah is the only woman he’s ever loved; but the couple is prepared to divorce in protest against any change to the law to accommodate same-sex marriage. “My wife and I, as […] MY wife and I just celebrated our 10-year anniversary. But later this year, we may be getting a divorce. In fact, my wife is the only woman I have ever loved, the mother of our children, my perfect match. So, the decision to divorce is not one we’ve taken lightly. And certainly, it’s not one that many will readily understand. And that’s because it’s not a traditional divorce. You see, after our divorce, we’ll continue to live together, hopefully for another 50 years. And, God willing, we’ll have more children. We’ll also continue to refer to each other as “husband” and “wife” and consider ourselves married by the Church and before God. So why do this? It will certainly complicate our lives as we try to explain our marital status on the sidelines during Saturday sport. The reason, however, is that, as Christians, we believe marriage is not a human invention. Our view is that marriage is a fundamental order of creation. Part of God’s intimate story for human history. Marriage is the union of a man and a woman before a community in the sight of God. And the marriage of any couple is important to God regardless of whether that couple recognises God’s involvement or authority in it. Which is why we are thinking of getting in touch with Simply the best Franklin Divorce Lawyer and getting all this done. No reason to wait either, we have made up our minds. My wife and I, as a matter of conscience, refuse to recognise the government’s regulation of marriage if its definition includes the solemnisation of same sex couples. The State (initially England) only got involved in marriage laws in 1753. For the 600 years before that in Europe, the Church acted as the official witness. Before the church had this role, marriage was simply a cultural norm ensuring children had the best possible upbringing. In Wales, for example, a couple would jump over a broomstick in the doorway of their new home to be married, and jump back across it to divorce. In fact, the main reason the State got involved at all was to address financial problems with property and inheritance law. This otherwise odd move of the State into marriage was ultimately permitted as long as it was seen as upholding a pre-existing societal good. Families, as the basic building block of communities, benefitted from the support and security of formal legislation. If our federal parliament votes to change the timeless and organic definition of marriage later on this year, it will have moved against the fundamental and foundational building block of Australian society and, indeed, human culture everywhere. Indeed, it raises a red flag when a government decides it is not content only having sovereignty over land, taxes and the military – but “words” themselves. This is why we are willing to divorce. By changing the definition of marriage, “marriage” will, in years to come, have an altogether different sense and purpose. It will not be about the mystery of difference in sexual unity, as children come from gendered dissimilarity. It will not be about building and securing communities into the future. When we signed that official-looking marriage certificate 10 years ago at Tuggeranong Baptist Church, we understood that the state was endorsing marriage, as currently defined, as the fundamental social institution – with all that this implied. But if this is no longer the case, then we no longer wish to be associated with this new definition. Marriage is sacred and what is truly “marriage” will only ever be what it has always been. It’s worth saying that our decision is not as extreme as it may seem. We will still benefit from the same tax and legal provisions of the state’s “de facto” laws. However, what is significant is this issue will echo the growing shift from state education to private religious institutions. This shift is no doubt because the majority of Australians, who are people of faith, believe their children are better served there. If the federal government pursues a change to the definition of marriage it will further alienate and divide the community. For example, there are many Christian denominations that will simply stop officiating for any civil marriages rather than go along with the government on this. Many Christians, like my wife and me, as well as people of other faiths, will simply reject the need for the State to recognise their marriage. Instead they will look to the authority of their church, mosque or temple. But there are broader implications for everyone, not just people of faith, to consider on this issue; for example, children’s rights, religious freedom, freedom of speech, and the broader fundamental rights of conscience and association. With our media’s relentless push to get this “over the line”, these issues have barely been noticed so far in the national debate. This has been a big decision for my wife and I. Some will accuse of us being bigoted or too hateful to share. But this couldn’t be further from our intentions. The truth is, “marriage” is simply too important. It is a sacred institution, ordained by God. It has always been understood to be that exclusive relationship where one man and one woman become “one flesh”. Any attempt to change the definition of marriage by law is not something in which we are able to partake. Here we stand, we can do no other, and I know we are not alone. Nick Jensen is the director of the Lachlan Macquarie Institute, which helps develop leaders in public policy (lmi.org.au) UPDATE: Nick’s brother Soren has penned a reply that bears reading. LIZ BURKE news.com.au THE Canberra man who says he’ll divorce his wife if gay marriage is legalised has warned of a marriage exodus among Christian couples who share his views. Nick Jensen told news.com.au he knew of a number of couples planning to legally divorce should the definition of marriage be changed to recognise same sex couples in Australia. In fact, the head of the Australian Christian Lobby group, for whom Mr Jensen has worked as a director, says he’s considering following suit and divorcing his own wife as well. “This is something that’s talked about very widely among churches,” Mr Jensen said. “I think it will have a significant impact on existing marriages, on future marriages, on young people decided whether they want to get married by the state.” Mr Jensen said he knew of couples who had made the same decision as he and his wife, Canberra GP Dr Sarah Jensen, announced in an article published by a Canberra magazine this week. Writing in Canberra CityNews, Mr Jensen said the couple’s decision to divorce was “not one they had taken lightly”. “If our federal parliament votes to change the timeless and organic definition of marriage later on this year, it will have moved against the fundamental and foundational building block of Australian society and, indeed, human culture everywhere... This is why we are willing to divorce,” he wrote. As well as indicating others would join the protest movement to abandon marriage, Mr Jensen told news.com.au a number of Christian groups were debating how to deal with potential changes, and that there were plans by marriage celebrants to hand in their licences “to avoid legal battles”. “In the last 48 hours there’s a story coming out of Idaho in the US with a couple of pastors there who carry out civil ceremonies and they’ve been told if they refuse to marry same sex couples they’d be put into jail,” he said. “This is a real concern for Christian’s who have their beliefs about marriage and what it might mean for them if same sex marriage is legalised.” Since his views were published on Wednesday, Mr Jensen has copped a torrent of criticism, with many of the hundreds of comments on this story labelling his decision “disgusting” and “bigoted”. But Mr Jensen told news.com.au he has also had a lot of support. “A lot of friends sent me a lot of love mail as well and my family’s been very supportive even it not all of them have the same line I do,” he said. “For me it comes down to what marriage is and this relationship between church and state, and the state is only a recent comer to this institution. This (protest) is just a natural consequence of a redefinition of marriage.” Mr Jensen said his decision to go public with his controversial views was to draw attention to the possibility of many couples wanting to “get out of marriage completely” as a consequence of gay marriage. The Canberra father-of-two has worked as a director with the Australian Christian Lobby and says the group supports his view. The group is publicly against same-sex marriage and encourages people to email MPs and senators to vocalise their objection under its “preserving marriage” campaign. The ACL’s managing director Lyle Shelton, who is publicly against same-sex marriage, told news.com.au he believed Mr Jensen had raised important issues, and that he himself would consider divorce for the same reason. “I would very much consider seriously what Nick’s saying,” he told news.com.au. “When you abolish terms like man and woman and husband and wife, then that of course affects the marriage contract that those of us were married under. It’s a bit like playing Aussie rules and half way those rules change to soccer. “My wife and I were married under a definition that said I’m a husband and my wife Wendy is a wife, and that means everything to us. To have those terms taken out of that, Bill Shorten doesn’t have the right to do that and that’s what he’s proposed.” A Canberra couple have vowed to get a divorce, ending their "sacred" 10-year union, if Australia allows same-sex couples to legally marry. Nick Jensen and his wife Sarah believe widening the definition of marriage to include same-sex couples threatens the sacred nature of the union and leaves the door open to polygamy. SHARE Share on Facebook SHARE Share on Twitter TWEET Link Divorce vow: Nick and Sarah Jensen. Photo: Facebook The Christian couple have been happily married for over a decade, have no intention of separating and hope to have more children. For all intents and purposes they have a healthy marriage. But in a novel protest against any successful move to legalise same sex-marriage in Australia, Mr Jensen wrote in an article published in Canberra CityNews on Wednesday that they are prepared to divorce. "My wife and I, as a matter of conscience, refuse to recognise the government's regulation of marriage if its definition includes the solemnisation of same sex couples," said Mr Jensen, who is director of the Lachlan Macquarie Institute, which partners with the Australian Christian Lobby to offer scholarships designed to develop a Christian worldview and foster leaders in government policy. Mr Jensen told Fairfax Media that he and his wife entered into their marriage "as a fundamental order of creation, part of God's intimate story for human history, man and woman, for the sake of children, faithful and for life". Advertisement "And so, if later on in the year the state does go ahead and changes the definition of marriage and changes the terms of that contract then we can no longer partake in that new definition unfortunately," he said. Legalising same-sex marriage would undermine "our most sacred institution, and have serious consequences for children who would grow up without a mother or father," Mr Jensen said. He also feared that recognising same-sex couples would mean that the definition of marriage could be expanded even further. "Once you say that marriage is detached from children, [that it's] just about love, then when three people come to the state and say 'well we're all in love', then the state has no grounds, except unjust discrimination, to say why they can't get married," he said. "When it becomes detached to the child's right to a mother and a father and the sacred institution that it is, then suddenly it becomes meaningless and those boundaries can't be put back in place," he said. The move is in stark contrast to high-profile celebrities who have vowed not to marry until gay marriage is legalised. While Brad Pitt and Angelina Jolie reneged on their promise not to marry in 2014, former Wallabies captain David Pocock and his partner Emma Palandri pledged in 2011 to not get married until same-sex marriage was legalised in Australia. LIZ BURKE news.com.au A CANBERRA couple has announced their intention to divorce if gay people are allowed to get married too. Nick Jensen, who posed with his wife Sarah on the cover of the latest issue of Canberra CityNews, writes of the Christian couple’s decision to end their marriage under the headline, “Gay law change may force us to divorce”. “My wife and I just celebrated our 10-year anniversary. But later this year, we may be getting a divorce,” he writes. “The decision to divorce is not one we’ve taken lightly. And certainly, it’s not one that many will readily understand. And that’s because it’s not a traditional divorce.” Mr Jensen goes on to explain the divorce plan, where the pair will continue to live together, have more kids, and refer to each other as husband and wife, but will legally end their marriage because they believe “marriage is not a human invention”. “Our view is that marriage is a fundamental order of creation. Part of God’s human history. Marriage is the union of a man and a woman before a community in the sight of God. And marriage of any couple is important to God regardless of whether that couple recognises God’s involvement or authority in it,” he writes. In the piece, Jensen describes the intervention of the state into marriage as “odd”, and says he and his wife refuse to recognise the government’s regulation of marriage if its definition includes same sex couples. “If our federal parliament votes to change the timeless and organic definition of marriage later on this year, it will have moved against the fundamental and foundational building block of Australian society and, indeed, human culture everywhere,” he writes. “Indeed, it raises a red flag when a government decides it is not content only having sovereignty over land, taxes and the military — but ‘words’ themselves. “This is why we are willing to divorce. By changing the definition of marriage, ‘marriage’ will, in years to come, have an altogether different sense and purpose.” The Canberra CityNews, an independent publication distributed in local businesses and published online, has faced comments from angry readers “appalled” by its decision to publish the piece. “This is absolutely disgusting, not to mention homophobic. I can’t believe such an article was published. You should be ashamed and you definitely owe to gay community an apology,” Natalie Sarah wrote on Facebook. “I hope this is a publicity stunt. If it isn’t then you massively shot yourself in the foot with this one Canberra CityNews,” Ross Kosub Garrett said on Facebook. The magazine’s editor, Ian Meikle, has defended his decision to publish the story, rejecting suggestions it was supporting a “homophobic” view. “I think this couple had an interesting angle, and that it was newsworthy,” he told news.com.au. “The article does not reflect the opinion of the paper. We published arguments and I decided it was a serious enough argument to genuinely warrant some attention. “It’s an unusual love story, and what would life be if people didn’t have different ways of life.” Mr Jensen is referred to in the article as director of the Lachlan Macquarie Institute and has previously been employed by the Australian Christian Lobby. The conservative lobby group is publicly against same sex marriage and encourages people to email MPs and senators to vocalise their objection under its “preserving marriage” campaign. Mr Jensen concluded his article saying he and his wife “know we are not alone” in their decision to divorce in protest. | Summary: Couples have been known to hold off on tying the knot until gay marriage is legal. A Christian couple in Australia, however, says they will divorce if gay marriage is allowed. "My wife and I just celebrated our 10-year anniversary. But later this year, we may be getting a divorce," Nick Jensen writes in a piece for the Canberra CityNews published yesterday. Why? "Marriage is the union of a man and a woman before a community in the sight of God" and "not a human invention," Jensen continues. If parliament votes to allow same-sex couples to marry, "then we no longer wish to be associated with this new definition." Should it come to divorce, Jensen and wife Sarah won't exactly part ways: They'll still live together, perhaps have more kids, use the terms "husband" and "wife," and "consider ourselves married by the Church and before God." They just won't be legally married. Jensen's piece has drawn plenty of strongly worded criticism, with some readers taking issue with the CityNews (which news.com.au describes as an "independent publication distributed in local businesses") for running it as a cover story. One commenter applauds the "genius" move to run the article, writing on Facebook, "It's going to do nothing but help the marriage equality cause. Now the country has international pressure not to be so stupid." While the editor of the CityNews says "the article does not reflect the opinion of the paper," he defends his decision to publish it. "I think this couple had an interesting angle, and that it was newsworthy," he tells news.com.au. Jensen himself, who says he's received plenty of "love mail," defends his stance to the Sydney Morning Herald, noting same-sex marriage would "have serious consequences for children who would grow up without a mother or father." Expanding the definition of marriage could also pave a path for polygamy, he says. | 3,554 | 424 | multi_news | en |
Summarize: The United Arab Emirates wants to build a mountain so the nation can control the weather—but some experts are skeptical about the effectiveness of this project, which may sound more like a James Bond villain’s diabolical plan than a solution to drought. The actual construction of a mountain isn’t beyond the engineering prowess of the UAE. The small country on the Arabian Peninsula has pulled off grandiose environmental projects before, like the artificial Palm Islands off the coast of Dubai and an indoor ski hill in the Mall of the Emirates. But the scientific purpose of the mountain is questionable. The UAE’s National Center for Meteorology and Seismology (NCMS) is currently collaborating with the U.S.-based University Corporation for Atmospheric Research (UCAR) for the first planning phase of the ambitious project, according to Arabian Business. The UAE government gave the two groups $400,000 in funding to determine whether they can bring more rain to the region by constructing a mountain that will foster better cloud-seeding. More Dubai Funds Jetpacks For Its Firefighters Last week the NCMS revealed that the UAE spent $588,000 on cloud-seeding in 2015. Throughout the year, 186 flights dispersed potassium chloride, sodium chloride and magnesium into clouds—a process that can trigger precipitation. Now, the UAE is hoping they can enhance the chemical process by forcing air up around the artificial mountain, creating clouds that can be seeded more easily and efficiently. “What we are looking at is basically evaluating the effects on weather through the type of mountain, how high it should be and how the slopes should be,” NCAR lead researcher Roelof Bruintjes told Arabian Business. “We will have a report of the first phase this summer as an initial step.” But some scientists don’t expect NCAR’s research will lead to a rain-inducing alp. “I really doubt that it would work,” Raymond Pierrehumbert, a professor of physics at the University of Oxford told Vocativ. “You’d need to build a long ridge, not just a cone, otherwise the air would just go around. Even if you could do that, mountains cause local enhanced rain on the upslope side, but not much persistent cloud downwind, and if you need cloud seeding to get even the upslope rain, it’s really unlikely to work as there is very little evidence that cloud seeding produces much rainfall.” Pierrehumbert, who specializes in geophysics and climate change, believes the regional environment would make the project especially difficult. “UAE is a desert because of the wind patterns arising from global atmospheric circulations, and any mountain they build is not going to alter those,” he said. Pierrehumbert concedes that NCAR is a respectable organization that will be able to use the “small amount of money to research the problem.” He thinks some good scientific study will come of the effort—perhaps helping to determine why a hot, humid area bordered by the ocean receives so little rainfall. But he believes the minimal sum should go into another project: “They’d be way better off putting the money into solar-powered desalination plants.” If the project doesn’t work out, at least wealthy Emirates have a 125,000-square-foot indoor snow park to look forward to in 2018. The Dubai skyline, with the Burj Khalifa illuminated in center. (Karim Sahib/AFP/Getty Images) The United Arab Emirates is certainly no stranger to ambitious mega-projects. Just take a look at the towering Burj Khalifa, the tallest building in the world, or the Palm Jumeirah, the artificial archipelago in the shape of a palm tree that juts out into the sea near Dubai. Even by the UAE’s standards, however, building a mountain would stand out as an ambitious plan. And perhaps what’s so remarkable about this plan is that despite its audacity, it would serve a sadly utilitarian purpose: to bring rain. According to the Dubai-based publication Arabian Business, the UAE is in the early stages of evaluating how a man-made mountain could help maximize rainfall in the country, consulting with experts from the U.S.-based National Center for Atmospheric Research (NCAR) to study the idea. “What we are looking at is basically evaluating the effects on weather through the type of mountain, how high it should be and how the slopes should be,” Roelof Bruintjes of NCAR told Arabian Business. “We will have a report of the first phase this summer as an initial step.” Mountains are important to rainfall. As moist air reaches a mountain it is forced to rise, cooling it. The air may then condense and turn to liquid, which can then fall as rain. This generally means that rainfall will occur on the mountain’s area that faces the wind while the other side of the mountain will be drier. Rain is a big issue in the UAE. Generally, it rains only a handful of days each year, and during the summer, when temperatures can reach 110 degrees Fahrenheit, there is often little or no rainfall at all. Most years the total annual rainfall doesn't break five inches — in comparison, Washington gets almost 40 inches a year. That can create a serious water security problem in places such as Dubai, designed to be an international destination for both work and play, and in rural areas where many farmers still rely on flood irrigation systems. Growing water consumption has already made UAE residents among the biggest per-capita consumers around the world, despite government attempts to restrict some water use. In response to this, for the past few years, there has been a campaign across the country to create more rainfall through artificially seeded clouds. Arabian Business recently reported that around $558,000 has been spent on 186 cloud seeding missions across the UAE last year, and the UAE Research Program for Rain Enhancement Science recently announced a $5 million research grant for teams studying the technology. So far, the campaign seems to have worked, with higher levels of rainfall than predicted. However, perhaps not everything has gone completely according to plan: A record rainfall in March, partially attributed to cloud seeding, included over 11 inches falling in less than 24 hours. That rainfall also created chaos in the country, with the heavy rains and winds resulting in flooding and canceled flights. Cloud seeding isn’t the only idea being pursued: The country has created a number of huge desalination plants to create fresh water from seawater. More ambitious (or, perhaps, unrealistic) ideas debated include a pipeline from Pakistan and icebergs floated down from the Arctic. Mountains can have a major effect on rainfall and could, in theory, at least be used to trap rainfall in a certain area. While the UAE does have some mountains in the far northern part of the country, only a small percentage of the population lives there. Therefore, the idea of building a mountain certainly sounds appealing, though it would probably be dependent on cost: One proposal to build a 1.2-mile-high mountain in the notoriously flat Netherlands was found to be feasible if the mountain were hollow. Estimates for the cost went as high as $230 billion. The UAE has spent $400,000 investigating the idea. Speaking to Arabian Business, NCAR’s Bruintjes acknowledged that the eventual cost of the project may be too much for the UAE. “Building a mountain is not a simple thing,” he said. More on WorldViews The UAE created a minister of happiness, but what does that even mean? China and the UAE give plenty of foreign aid, but the details can be hard to nail down Starting in 1996, Alexa Internet has been donating their crawl data to the Internet Archive. Flowing in every day, these data are added to the Wayback Machine after an embargo period. | Summary: Even with a major downturn in the oil business, the United Arab Emirates still has enough money to look at a few modest construction projects-including building a mountain in the desert. The Gulf nation, which is desperately short of rain, has paid $400,000 to the Colorado-based University Corporation for Atmospheric Research to study the possibility of boosting rainfall with an artificial mountain, Arabian Business reports. The UAE spent $558,000 on cloud-seeding, a process meant to increase the amount of rain produced by clouds, last year alone; lead researcher Roelof Bruintjes explains that a mountain would cause air to rise, creating more clouds to seed, though they're not entirely sure how tall the mountain should be. "Building a mountain is not a simple thing," he says. The next step will be to submit the team's plan to an engineering firm. Experts seem to think the idea is as outlandish as it sounds. "I really doubt that it would work," Oxford physics professor Raymond Pierrehumbert tells Vocativ. "You'd need to build a long ridge, not just a cone, otherwise the air would just go around," he explains, adding that even if that was possible, under local conditions, "it's really unlikely to work as there is very little evidence that cloud seeding produces much rainfall." He says no mountain will change the fact that the area is a desert, and the UAE would be far better off "putting the money into solar-powered desalination plants." Exactly how much money the project would cost isn't clear, though the Washington Post notes that a plan to give the Netherlands its first tall mountain had an estimated price tag of up to $230 billion-and was only found to be feasible if the mountain was hollow. | 1,765 | 393 | multi_news | en |
Summarize: RELATED APPLICATION This application is a continuation in part of applicant's copending application Ser. No. 06/921,218, filed Oct. 20, 1986, now U.S. Pat. No. 4,925,664, entitled "Spider Toxins and Methods for their Use as Blockers of Calcium Channels and Amino Acid Receptor Function," which application is incorporated herein by reference. BACKGROUND 1. The Field of the Invention The present invention generally relates to the isolation of certain toxins from spider venoms and the use of those toxins as inhibitors of the functions of ion channels. In particular, the present invention relates to spider venom toxins and their use as blockers of calcium channels in the central nervous and neuromuscular systems of organisms, including humans. 2. The Background of the Invention Movement of calcium ions across cell membranes is a critically important event in the normal functioning of excitable tissues such as vascular smooth muscle, cardiac muscle, and the central nervous system. Influx of calcium ions through specialized channels in the cell membranes regulates release of substances such as hormones and neurotransmitters. The movement of calcium ions also regulates contraction of heart muscle and of vascular smooth muscle in the wall of blood vessels. Abnormal influx of calcium ions has been reported to play a role in the pathogenesis of various cardiovascular disorders (e.g., anoxic/ischemic heart disease), and drugs capable of blocking the movement of calcium through calcium channels have been used for treatment of cardiac arrhythmias, coronary artery disease, and cardiomyopathy. The currently used drugs, however, have non-specific physiological effects and varying tissue specificities that can lead to undesirable side-effects in patients. Moreover, there are several known subtypes of calcium channels with varying physiological actions and no drug that specifically blocks certain of these subtypes is known. In the nervous system, calcium influx into the presynaptic nerve terminal via calcium channels is a necessary prerequisite for the release of chemical neurotransmitter at synapses and thus for the proper functioning of these synapses. Lowering of the extracellular calcium concentration is routinely used by neurophysiologists to reduce or abolish synaptic transmission in isolated pieces of nervous tissue. It has not been possible, however, to specifically affect synaptic transmission in vivo in the central nervous system ("CNS") by manipulating the function of neuronal calcium channels With the exception of the omega-conotoxin recently isolated from the venom of the marine snail Conus geographus, no drug with sufficiently specific or potent effects on CNS calcium channels is known. Abnormal influx of calcium is thought to be very important in the pathogenesis of several CNS disorders, including anoxic/ischemic (stroke) damage, epilepsy, and the neuronal death associated with chronic epilepsy. Again, the paucity of chemical agents that potently and specifically block CNS calcium channels has impeded the development of an effective drug therapy for these prevalent neurological problems. Thus, it would be a very considerable improvement in the art if it were possible to develop chemical agents that specifically and potently block calcium channel function in the CNS. In particular, it would be an advancement in the art to provide a specific blocker for particular subtypes of calcium channel. Similarly, it would be an advancement in the art to provide a specific blocker of calcium channels in the CNS. Such chemical compositions and methods for their use are disclosed and claimed below. BRIEF SUMMARY AND OBJECTS OF THE INVENTION The present invention is related to the isolation, identification, and use of spider venoms and toxins contained within these venoms. In particular, the present invention is related to the isolation and use as calcium channel blockers of certain toxins from spider venom. As discussed above, calcium channels are intimately involved in the functions of the cardiovascular system since calcium influx affects contraction of cardiac muscle and vascular smooth muscle. Similarly, calcium influx into nerve cells is required for the release of chemical neurotransmitter substances at synapses and, therefore, for the normal functioning of the nervous system. Calcium influx into nerve cells is also involved in mediating certain electrical responses of those cells. Abnormal calcium influx into cells is associated with serious cardiovascular and neurological disorders. The present invention is related to obtaining toxins from spider venoms, which toxins have specific and potent blocking effects on calcium channels within the organism. Within the scope of the present invention, spider venom is obtained by milking spiders of various species. That is, the spider venom is obtained by electrical stimulation of the spider to cause release of the venom and subsequent suction in order to collect the released venom. This assures that impurities, which have traditionally been contained within spider venoms obtained by conventional techniques, are eliminated. Spider venoms are known to be a complex mixture of enzymes, peptide toxins, nucleotides, free amino acids, and other molecules. As a result, in order to obtain useful spider toxins it is necessary to separate the various components of the whole spider venom. According to one embodiment of the present invention, whole venoms are fractionated by gel filtration to separate components of the venom by relative molecular mass. It will be appreciated, however, that any type of fractionation technique or other technique may be useful to obtain the spider venom toxins necessary for use in the present invention. A group of specific spider venoms has been isolated and used extensively in the context of the present invention. The spiders that have been used within the scope of the present invention are Agelenopsis aperta spiders. Agelenopsis spiders are members of the funnel-web grass spider family Agelenidae and are commonly found in meadows and other grassy areas within the western United States. The primary specific toxin which falls within the scope of the present invention has been isolated from the Agelenopsis spider. In particular, a relatively high molecular weight toxin that suppresses synaptic transmission in the vertebrate central nervous system by blocking calcium channels. For ease of identification this toxin will be sometimes generally referred to as "AG1" during the present description of the invention. AG1 has been identified by amino acid sequencing techniques as a 48 amino acid peptide. The toxin is found to have the following sequence: ##STR1## The composition has a molecular weight of approximately 5274. The gene which produces the AG1 toxin has been identified by employing a new procedure. The procedure uses a "polymerase chain reaction" (PCR) conducted under conditions which have recently been identified in the relevant literature. See, R. K. Saiki et al., Science 230, p. 1350 (1985); Saiki et al., "Primer-Directed Enzymatic Amplification of DNA with a Thermostable DNA Polymerase," Science 239, 487 (January 1988), which are collectively incorporated herein by this reference. In order to clone the toxin gene two oligonucleotide primers were prepared. The first primer was specific only for the "poly A" tail of the mRNA. The second oligonucleotide primer was engineered from the cDNA of the coding gene sequence. All of the mRNA in the RNA population was reverse transcribed to cDNA using the d(T) tailed primer. The specificity in the procedure comes with the amino terminal primer which is designed specifically from the protein sequence. Thus, only the cDNA which contains this sequence will be efficiently amplified in this system, as described in Saiki, et al. The analysis of the anchored PCR products obtained from the reverse transcribed Agelenopsis venom gland mRNA are shown in FIG. 2, as described in greater detail below. Southern hybridization with the internal probe confirmed the correct gene sequence. DNA sequencing of this anchored PCR product confirmed that it contained the complete coding sequence of AG1, as well as all DNA sequences preceding the poly (A) tail. The sequence of the gene is set forth in Table 4 below. Analysis of the translated peptide sequence indicated a protein having a molecular weight of approximately 5,280 daltons, with 8 cysteine residues. This data is set forth below in Table 5. This data indicates that the gene sequence identified is that which is responsible for the production of AG1 in the Agelenopsis aperta spider. Identification of the gene responsible for the production of AG1 facilitates the production of the toxin by using i.e., recombinant DNA techniques, and may result in the ability to genetically engineer higher plants with the ability to produce the toxin. It has been found that AG1 produces blockade of synaptic transmission under certain conditions without affecting axonal conduction of action potentials. The AG1 toxin affects transmission of the nerve impulse across the synapse. AG1 is also found to block transmission in certain central nervous system cells by blocking calcium currents. It is particularly noteworthy that AG1 is not acutely toxic to the cells tested and does not affect the electric excitability of the neurons themselves. Thus, this suggests that AG1's effects are not produced by acute cytotoxic action. Simply stated, CNS transmission is blocked without damaging the cells involved. It is a primary object of the present invention to provide calcium channel blockers and methods for their use which have specific and identifiable effects on an organism. Another object of the present invention is to provide a specific calcium channel blocker which affects the central nervous system. It is another object of the present invention to provide calcium channel blockers for use as research tools and for use in the clinical setting. It is also an object of the present invention to identify the amino acid sequence of the AG1 toxin responsible for blocking synaptic transmission in the central nervous system. It is a similar object of the present invention to isolate and identify the gene responsible for production of the AG1 toxin that blocks synaptic transmission. These and other objects of the present invention will become apparent upon reading the following detailed description and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic drawing representing the procedure employed in identifying and isolating the gene responsible for the production of AG1. FIG. 2 is an autoradiograph of the southern analysis of the reaction products confirming the isolation and identification of the gene responsible for the production of AG1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As discussed above, the present invention is related to new and unique calcium channel blockers, methods for their isolation, and methods of application of such molecules. In particular, the present invention relates to the use of isolated toxins obtained from spider venom for use as specific calcium channel blockers. It has been found within the scope of the present invention that certain spider venoms may selectively act on the central nervous system. More particularly, it has been found that spider venoms can have specific activities on calcium channels within the organism. An additional benefit of the present invention is that the isolated toxins act without significant cytotoxicity. Thus, the toxins do not block channels by destroying the cells within the systems in which they are active. Additionally, the toxins of the present invention generally act without affecting axonal conduction within the nervous system. It will be appreciated, therefore, that only calcium channels are affected by the toxin that acts on the central nervous system. I. Techniques for Isolation of Venoms In order to avoid impurities within the spider venom and the isolated toxins, the spider venom which was used for the tests described below was electrically milked from the spiders using a method which employs safeguards to prevent contamination of the venom by abdominal regurgitate or hemolymph. Once the spider venom is obtained by electrical milking techniques, it is further purified using gel filtration chromatography or other similar related techniques. In addition, it is frequently desirable for final fractionation of the spider venom to be performed by high performance liquid chromatography ("HPLC"). Thus, using the technique of electrically milking the spider coupled with gel filtration chromatography and high performance liquid chromatography it is possible to obtain purified and usable spider toxins. It will be appreciated, however, that other equivalent techniques may also be employed the spider toxins used. II. Specific Toxin within the Scope of the Invention While it will be appreciated that additional toxins may also fall within the scope of the present invention, the following relates to the identification and isolation of a specific toxin which has been found to have the characteristics required for a usable calcium channel blocker as described above. In addition, the native gene responsible for the production of the toxin has been identified. Using the techniques described above relating to the collection of venom, a toxin has been isolated from the Agelenopsis aperta spider having a molecular weight of approximately 5274 and the peptide sequence described herein. It has been found that AG1 blocks synaptic transmission in in vitro preparations of chick cochlear nucleus and rat hippocampus. In experiments performed using the toxin, it has been found that the toxin is very potent in that 100 nanomolar concentrations produce half-maximol blockade of synaptic transmission in rat hippocampus slices and 1 micromolar concentrations produce complete blockade. Complete blockade using AG1 on central nervous system cells occurs in the absence of presynaptic action potentials and the time course of action is unaffected by the rate of presynaptic stimulation. Partial blocks achieved by brief exposure to dilute AG1 toxin were stable for at least one hour and were unaffected by increases or decreases in stimulation rate. Partial blockade by AG1 however, can be largely reversed by increasing the extracellular calcium concentration. Subsequent reduction in calcium concentration, however, causes the postsynaptic response to decline to its previous level of partial blockade. In the absence of toxin, the same increase in calcium has no effect on the amplitude of responses. These results indicate that this toxin acts on calcium channels to produce long lasting blockade of transmission. The effects are independent of stimulation frequency, suggesting that the toxin does not act primarily on synthesis or reuptake of transmitter. Calcium antagonism of the effects makes it improbable that the toxin causes massive release of transmitter. In summary, it is found that this toxin is antagonized by increasing calcium concentrations and produces blockade of synaptic transmission in cochlear chick nucleus and rat hippocampus without affecting axonal conduction. In addition, this toxin has been found not to be acutely toxic and does not affect the electrical excitability of cochlear nucleus neurons themselves, indicating that its effects are not produced by acute cytotoxic action. III. Comparison with other Calcium Channel Blockers Receptor- and voltage-activated calcium channels are of fundamental importance in the survival and function of virtually all cell types. Entry of calcium through such channels regulates a variety of cellular activities including contraction of cardiovascular muscle and the release of neurotransmitters from nerve cells. There are presently three major known classes of organic calcium channel blockers, as opposed to inorganic blockers such as manganese or lanthanum. These organic calcium channel blockers include: phenylalkylamines such as verapamil; benzothiazepines such as diltiazem; and dihydropyridines such as nifedipine. The currently available organic calcium channel blockers have pronounced actions on heart and vascular smooth muscle, although relative selectivity for these two types of tissues varies among these compounds. A second notable feature of these agents is that, although they will bind to brain tissue, they have either no effect or a relatively minor effect on the function of neurons in the central nervous system, particularly as compared to their striking effects on heart and vascular smooth muscle. The AG1 toxin derived from Agelenopsis aperta venom has properties that very clearly distinguish it from the currently available calcium channel blockers. AG1 acts primarily, if not exclusively, on neuronal calcium channels as opposed to heart or vascular smooth muscle calcium channels. This tissue selectivity is opposite to that seen in the compounds mentioned above. Because of the importance of calcium and calcium channels to the function of neurons, there are a variety of potential applications of compounds within the scope of the present invention. Calcium influx through channels mediates neurotransmitter release and modulates neuronal excitability. Selective blockers of neuronal calcium channels, therefore, could modify neuronal excitability by effects on both presynaptic and postsynaptic calcium channels. Accordingly, appropriate calcium channel blockers could be used in treatment of several neurological disorders that are thought to involve excessive neuronal excitation: e.g., stroke, traumatic head injury, epilepsy, and neurodegenerative disorders such as Huntington's disease and Alzheimer's disease. IV. Amino Acid Sequencing AG1 was further analyzed in order to determine its amino acid sequence. Initially, venom was obtained from Agelenopsis aperta spiders using the techniques described herein. Active fractions of the venom were pooled and subjected to separation by ion-exchange chromatography. The fraction of the venom constituting the AG1 toxin was then isolated using the techniques described herein, including in Example 2. The AG1 toxin was then analyzed by employing known techniques and with the aid of a mechanical amino acid analyzer. The results of the amino acid sequence analyses of AG1 yielded a 48 amino acid peptide. The peptide has a molecular weight in the range of from approximately 5272 to approximately 5282 daltons. The sequence of the peptide as identified by the procedure set forth above is as follows: ##STR2## V. Identification of the Gene Responsible for Production of AG1 Employing the amino acid sequence data obtained as described above, the gene responsible for the production of AG1 was isolated and identified. Initially, the possible codons responsible for each amino acid in the sequenced peptide were identified and plotted. Table 1 lists the possible codons which relate to each amino acid in the peptide. It will be appreciated that the possible number of sequences available to produce the peptide is very large, due to the fact that some of the amino acids can be produced by up to six (6) different nucleotide sequences. When it is appreciated that a 48 amino acid peptide is involved, with most of the amino acids possibly being encoded by multiple nucleotide sequences, it will be appreciated that sequential testing using possible oligonucleotide probes was found to be impractical. TABLE 1 AG1 IN ALL CODONS. 5 10 15 20 GLU ASP ASN CYS ILE ALA GLU ASP TYR GLY LYS CYS THR TRP GLY GLY THR LYS CYS CYS GAA GAC AAC TGT ATT GCT GAA GAC TAC GGT AAG TGT ACT TGG GGT GGT ACT AAG TGT TGT GAG GAT AAT TGC ATC GCC GAG GAT TAT GGC AAA TGC ACC GGC GGC ACC AAA TGC TGC ATA GCG GGA ACA GGA GGA ACA GCA GGG ACG GGG GGG ACG 25 30 35 40 ARG GLY ARG PRO CYS ARG CYS SER MET ILE GLY THR ASN CYS GLU CYS THR PRO ARG LEU AGA GGT AGA CCA TGT AGA TGT TCT ATG ATT GGT ACT AAC TGT GAA TGT ACT CCA AGA TTG GGC CCT TGC TGC TCC ATC GGC ACC AAT TGC GAG TGC ACC CCT CTA AGG GGA AGG CCG AGG TCA ATA GGA ACA ACA CCG AGG TTA CGT GGG CGT CCC CGT TCG GGG ACG ACG CCC CGT CTG CGC CGC CGC AGT CGC CTT CGA CGA CGA AGC CGA CTC CGG CGG CGG CGC 45 50 55 60 ILE MET GLU GLY LEU SER PHE ALA ATT ATG GAA GGT TTG TCT TTC GCT ATC GAG GGC CTA TCC TTT GCC ATA GGA TTA TCA GCG GGG CTG TCG GCA CTT AGT CTC AGC Accordingly, a technique employing PCR was developed and is illustrated schematically in FIG. 1. PCR is well documented in the literature, including the citations set forth above. Essentially PCR allows the production of a selected DNA sequence when the two terminal portions of the sequence are known. Primers, or oligonucleotide probes, are obtained which correspond to each end of the sequence of interest. Using PCR, the central portion of the DNA sequence is then synthetically produced. In sequencing the gene corresponding to AG1, a first primer was selected which corresponds to the poly (A) terminus, also sometimes referred to herein as a d(T) tailed primer. The d(T) tailed primer also includes a restriction site downstream from the d(T) segment. In this particular procedure a NotI restriction site was included (CGCGGCCGC). The d(T) tailed primer is illustrated at 1 in step (a) of FIG. 1, and the mRNA of the spider is illustrated at 2. All mRNA is then reverse transcribed to cDNA using the d(T) tailed primer as is represented graphically by the arrow in step (a). The synthetic cDNA is then denatured and annealed as is shown in step (b). At this point an appropriate NH2 primer or oligonucleotide is synthesized. See, Table 2. In the synthesis of the primer the data of Table 1 is employed to determine the possible sequences which may be usable. In addition, in order to select the sequences which are most likely to result in the actual sequence, preference data of the type illustrated in Table 3 is employed. At this point, the primer 3 is bound to the denatured cDNA and a cDNA sequence is produced as illustrated at step (c) in FIG. 1 Because the primer is specific to the desired mRNA sequence, only the cDNA which corresponds to the chosen sequence will be effectively amplified. The resulting material is amplified over multiple cycles, as is taught in PCR procedure as is shown at step (d). In this particular case amplification took place over 39 cycles in order to assure maximum amplification. The result of the process is a synthetic cDNA sequence which corresponds to that of the gene of interest. This is illustrated at (e). The DNA produced is then digested with the appropriate restriction enzymes and cleaved at the engineered restriction site. (Cleaving the restriction site enables it to be cloned). This step is illustrated at (f). TABLE 2__________________________________________________________________________Primers for PCR amplification of AG1 Gene Sequnces AA #: 1 2 3 4 5 6 7 8__________________________________________________________________________Amino acid: E D N C I A E DAll possible codons: GAG GAC AAC TGT ATT GCA GAG GA GAA GAT AAT TGC ATC GCC GAA ATA GCG GCTNH--K Oligo: GAG GAC AAC TGC ATT GCA GAG GA GAT AAT ATC GCC GAA GCG GCT__________________________________________________________________________ TABLE 3__________________________________________________________________________ Drosophila Preference (1)A.A. # of codons Favored codons h = high bias gene l = low bias gene Human Preference(2)__________________________________________________________________________C 2 UGC h = 0.97 l = 0.65 0.70D 2 GAC h = 0.61 l = 0.22 0.62E 2 GAG h = 0.91 l = 0.64 0.60F 2 UUC h = 0.94 l = 0.57 0.65H 2 CAC h = 0.86 l = 0.57 0.58K 2 AAG h = 0.97 l = 0.60 0.55N 2 AAC h = 0.94 l = 0.44 0.66Q 2 CAG h = 0.99 l = 0.56 0.74Y 2 UAC h = 0.89 l = 0.52 0.53I 2 AUC h = 0.75 l = 0.32 0.64 AUU h = 1.00 l = 0.74 0.87A 4 GCC h = 0.94 l = 0.71 0.71 GCUG 4 GGC h = 0.75 l = 0.64 0.59 GGUP 4 CCC h = 0.84 l = 0.52 0.65 CCAT 4 ACC h = 0.76 l = 0.40 0.47 ACG h = 0.81 l = 0.63 0.59V 4 GUC h = 0.85 l = 0.68 0.77 GUGL 6 CUG h = 0.71 l = 0.34 0.46 CUC h = 0.83 l = 0.49 0.68R 6 CGC h = 0.95 l = 0.37 0.28 CGUS 6 UCC h = 0.65 l = 0.45 0.33 UCG AGC h = 0.84 l = 0.62 0.62__________________________________________________________________________ (1)Sharp, P. et al., Nucl. Acids Res. 16, 8207 (1988). (2)Lathe, R. J. Mol. Biol. 183, 1 (1985). Finally, the produced cDNA sequence can be cloned into an appropriate vector using conventional techniques, analyzed and sequenced. The DNA sequence is presented in Table 4. A direct amino acid translation of this anchored PCR product revealed that it corresponded to the complete coding sequence for AG1, as well as all DNA sequences preceding the poly (A) tail. See, Table 5. The exact function and purpose of the portion of the gene between the stop codon for the peptide and the beginning of the poly (A) tail is not known at this time. The complete sequence is set forth in Table 4. Table 4 shows the DNA sequence above the corresponding peptide for each of the 48 peptides. Table 4 also sets forth the DNA sequence between the end of the material useful for encoding the peptide and the beginning of the poly (A) tail, as well as the tail itself. Additional detail concerning the procedure described above is included herein in Example 3. FIG. 2 illustrates the results of Southern analysis of one-tenth of the reaction products. "A" is an autoradiograph of an ethidium stained gel of one-tenth of the reaction products obtained from the amplification of 25 ng mRNA using the amino primer d(T) tailed primer adapter. The size marker lane was Hinf I digested φ174 DNA. "B" is the results of Southern analysis of the reaction products confirmed by probing with an internal gene sequence. The results show dramatically the effectiveness of the technique described above. TABLE 4__________________________________________________________________________Translated SequenceSequence Range: 1 to 317 ##STR3## ##STR4## ##STR5## ##STR6## ##STR7## ##STR8## ##STR9##ATTTCTAGAGTTCGTCTTATCGCTACCTCGTCTTGTAGATCAAATGAATGT ##STR10##TAATATAATTATAATATAATTTATTAGGAGTTTATTCCTACATATTAAAAC ##STR11##ACTACTTTAATTTTTAAGAAATAAGCGTTTTTTTTT TTTTTTTTTTTTTTT ##STR12##TTTTTTTTTTT__________________________________________________________________________ TABLE 5______________________________________peptideK AA Composition DataCalculated Molecular Weight = 5280.729Estimated pI = 6.465Amio Acid Composition: Number Percent______________________________________Non-polar:Ala 2 4.17Val 0 0.00Leu 2 4.17Ile 3 6.25Pro 2 4.17Met 2 4.17Phe 1 2.08Trp 1 2.08Polar:Gly 6 12.50Ser 2 4.17Thr 4 8.33Cys 8 16.67Tyr 1 2.08Asn 2 4.17Gln 0 0.00Acidic:Asp 2 4.17Glu 4 8.33Basic:Lys 2 4.17Arg 4 8.33Bis 0 0.00______________________________________ Recombinant Expression Provision of a suitable DNA sequence encoding the desired protein permits the production of the protein using recombinant techniques now well known in the art. The coding sequence can be obtained by retrieving a cDNA or genomic sequence from a native source of the protein or can be prepared synthetically using the accurate amino acid sequence from the nucleotide sequence of the gene. When the coding DNA is prepared synthetically, advantage can be taken of known codon preferences of the intended host. Expression systems containing the requisite control sequences, such as, promoters, and preferably enhancers and termination controls, are readily available and known in the art for a variety of hosts. Thus, the desired proteins can be prepared in both procaryotic and eucaryotic systems, resulting, in the case of many proteins, in a spectrum of processed forms. The most commonly used procaryotic system remains E. coli, although other systems such as B. subtillis and Pseudomonas could also be used. Suitable control sequences for procaryotic systems include both constitutive and inducible promoters including the lac promoter, the trp promoter, hybrid promoters such as tac promoter, and the lambda phage P 1 promoter. In general, foreign proteins may be produced in these hosts either as fusion or mature proteins; when the desired sequences are produced as mature proteins, the sequence produced may be preceded by a methionine which is not necessarily efficiently removed. Accordingly, the peptides and proteins claimed herein may be preceded by an N-terminal Met when produced in bacteria. Moreover, constructs may be made wherein the coding sequence for the peptide is preceded by an operable signal peptide which results in the secretion of the protein. When produced in procaryotic hosts in this matter, the signal sequence is removed upon secretion. A wide variety of eucaryotic hosts is also now available for production of recombinant foreign proteins. As in bacteria, eucaryotic hosts may be transformed with expression systems which produce the desired protein directly, but more commonly signal sequences are provided to effect the secretion of the protein. Eucaryotic systems have the additional advantage that they are able to process introns which may occur in the genomic sequences encoding proteins of higher organisms. Eucaryotic systems also provide a variety of processing mechanisms which result in, for example, glycosylation, oxidation or derivatization of certain amino acid residues, conformational control, and so forth. Commonly used eucaryotic systems include yeast, insect cells, mammalian cells, avian cells, and cells of higher plants. The list is not exhaustive. Suitable promoters are available which are compatible and operable for use in each of these host types as well as are termination sequences and enhancers. As above, promoters can be either constitutive or inducible. For example, in mammalian systems, the MTII promoter can be induced by the addition of heavy metal ions. The particulars for the construction of expression systems suitable for desired hosts are well known to those in the art. For recombinant production of the protein, the DNA encoding it is suitably ligated into the expression system of choice, and the system is then transformed into the compatible host which is then cultured and maintained under conditions wherein expression of the included gene takes place. The protein thus produced is recovered from the culture, either by lysing the cells or from the culture medium as appropriate. A "mutation" in a protein alters its primary structure (relative to the commonly occurring or specifically described protein) due to changes in the nucleotide sequence of the DNA which encodes it. These mutations specifically include allelic variants. Mutational changes in the primary structure of a protein result from deletions, additions, or substitutions. Such changes involving only 3 or less amino acid residues are generally preferred. A "deletion" is defined as a polypeptide in which one or more internal amino acid residues are absent. An "addition" is defined as a polypeptide which has one or more additional internal amino acid residues as compared to the wild type. A "substitution" results from the replacement of one or more amino acid residues by other residues. A protein "fragment" is a polypeptide consisting of a primary amino acid sequence which is identical to a portion of the primary sequence of the protein to which the polypeptide is related. Preferred "substitutions" are those which are conservative, i.e., wherein a residue is replaced by another of the same general type. As is well understood, naturally-occurring amino acids can be subclassified as acidic, basic, neutral and polar, or neutral and nonpolar. Furthermore, three of the encoded amino acids are aromatic. It is generally preferred that encoded peptides differing from the native form contain substituted codons for amino acids which are from the same group as that of the amino acid replaced. Thus, in general, the basic amino acid Lys, Arg, and His are interchangeable; the acidic amino acids aspartic and glutamic are interchangeable; the neutral polar amino acids Ser, Thr, Cys, Gln, and Asn are interchangeable; the nonpolar aliphatic acids Gly, Ala, Val, Ils, and Leu are conservative with respect to each other (but because of size, Gly and Ala are more closely related and Val, Ile and Leu are more closely related), and the aromatic amino acids Phs, Trp, and Tyr are interchangeable. While proline is a nonpolar neutral amino acid, it represents difficulties because of its effects on conformation, and substitutions by or for proline are not preferred, except when the same or similar conformational results can be obtained. Polar amino acids which represent conservative changes include Ser, Thr, Gln, Asn; and to a lesser extent, Met. In addition, although classified in different categories, Ala, Gly, and Ser seem to be interchangeable, and Cys additionally fits into this group, or may be classified with the polar neutral amino acids. Some substitutions by codons for amino acids from different classes may also be useful. Because recombinant materials for the proteins of the invention are provided, these proteins can be made recombinantly. Because of the variety of post-translational characteristics conferred by various host cells, various modifications for the naturally-occurring proteins will also be obtained. A "modified" protein differs from the commonly occurring protein as a result of post-translational events which change the glycosylation or lapidation pattern, or the primary, secondary, or tertiary structure of the protein. It should be further noted that if the proteins herein, such as AG1, are made synthetically, substitutions by amino acids which are not encoded by the gene may also be made. Alternative residues include, for example, the ωamino acids of the formula H 2 N(CH 2 ) n COOH wherein n is 2-6. These are neutral, nonpolar amino acids, as are sarcosine (Sar), t-butylalanine (t-BuA), t-butylglycine (t-BuG), N-methyl Ila (N-MeIle), and norleucine (Nle). Phenylglycine, for example, can substituted for Trp, Tyr or Phe an aromatic neutral amino acid; citrulline (Cit) and methionine sulfoxide (MSO) are polar but neutral, cyclohexyl alanine (Cha) is neutral and nonpolar, cysteic acid (Cya) is acidic, and ornithine (Orn) is basic. The conformation conferring properties of the proline residues may be obtained if one or more of these is substituted by hydroxyproline (Hyp). V. EXAMPLES The following examples are given to illustrate particular compositions and methods within the scope of the present invention but they are not intended to limit the scope of the present invention. EXAMPLE 1 A spider toxin within the scope of the present invention was isolated from the Agelenopsis aperta spider. Spider venom was obtained from, and species identification provided by, Spider Pharm, Inc. of Black Canyon City, Ariz. and Natural Product Sciences, Inc., Salt Lake City, Utah. Agelenopsis aperta spiders were electrically milked using a method that employs safeguards to prevent contamination of venom by abdominal regurgitate or hemolymph. Venom was diluted 1 to 10 with avian Tyrode solution (140 mM NaCl, 4 mM KCl, 4 mM NaHCO 3, 1 mM MgSO 4, 3 mM CaCl, 1.2 mM NaH 2 PO 4, 10 mM HEPES, 10 mM glucose) and fractionated by gel filtration using Bio-Gel P-10 and a 0.7×30 cm column and collected in 0.5 ml fractions. These fractions were assayed for blockade of synaptic below. HPLC separation of active gel filtration fractions was performed using a Vydac C-18 reverse phase column. Components were eluted from the column over a period of 60 minutes using a 0-60% linear gradient of 60% acetonitrile in 0.1% trifluoroacetic acid. Elution was monitored by absorbance detection at 214 nm. Peaks were collected manually, dried down, stored at -20° C., and then reconstituted with avian Tyrode before use. For gel electrophoresis, a 15 ul sample of each gel filtration fraction was mixed with 7.5 ul of 3X sample buffer (18% 1M tris-HCL, pH 6.8, 15% 2 mercaptoethanol, 30% glycerol, 7% sodium dodecyl-sulfate, 0.001% bromphenol blue) and the entire sample was loaded onto a 10% to 20% gradient polyacrylamide gel using the slab method. Electrophoresis was performed at 20 watts constant power for three hours. Gels were stained with Coomassie blue. The toxin so isolated had a molecular weight of approximately 6,000 as estimated from SDS polyacrylamide gels as was designated AG1. The toxin was bath-applied to cochlear nucleus neurons in an in vitro preparation of chick brain stem. Upon stimulation of the cochlear nerve innervating the cochlear nucleus, it was found that the toxin blocked transmission between the cochlear nerve afferents and the cochlear nucleus neurons. EXAMPLE 2 Agelenopsis aperta spiders were electrically milked using a method that employs safeguards to prevent contamination of venom by abdominal regurgitate or hemolymph. Venom was diluted to 1 to 10 with avian Tyrode solution and fractionated by gel filtration using Bio-Gel P-10 and a 0.7×30 cm column and collected in 0.5 ml fractions. These fractions were assayed for blockage of synaptic transmission using an in vitro preparation of the chick cochlear nucleus. Active fractions (fractions 7-9) were pooled and subjected to separation by ion-exchange chromatography using NaCl concentrations of 10-300 mM, 0.5 ml fractions were again collected and assayed. A fraction eluting in 70 mM NaCl contained the material active in blocking synaptic transmission in the chick cochlear nucleus. This fraction was then subjected to separation by high-performance liquid chromatography (HPLC) using a Vydac C-18 reverse-phase column. Components were eluted from the column over a period of 60 minutes using a 0-60% linear gradient of 60% acetonitrile in 0.1% trifluorooacetic acid. Elution was monitored by absorbance detection at 214 nm. Peaks were collected manually, dried down, stored at -20° C. and then reconstituted in avian Tyrode solution before being re-assayed on the chick cochlear nucleus preparation. A single major peak in the chromatogram appeared to contain most of the activity in the AG1 fraction; this peak was subjected to amino acid composition and sequence analyses using the following methods. Samples of AG1 were subjected to amino acid analysis by known techniques. The samples were hydrolyzed in vacuo with 6N HCL, at 105° C. for 24 hours. Acid Was removed by drying in a Speed-Vac and the residue was analyzed by ion-exchange chromatography on a Beckman Model 121 amino acid analyzer. Other samples of AG1 were reduced, carboxymethylated, and analyzed in a Beckman 890D spinning cup peptide sequencer using a 0.1M Quadrol program. Polybrene carrier was precycled by running four steps in the presence of 100 nmol of Ser-Gly. PTH-amino acids were analyzed by HPLC using methods described by Gray et al. (J. Biol. Chem. 256:4734-4740, 1981). The results of amino acid and sequence analyses of AG1 yielded the following 48-amino acid peptide: ##STR13## EXAMPLE 3 The coding gene for AG1 from Agelenopsis was isolated. The procedure for isolating the gene is outlined as follows: 1 Isolate RNA from spider; purify mRNA. 2. Synthesize oligonucleotide primer corresponding to the amino-terminal protein sequence data and internal primer if data available. 3. Reverse transcribe the RNA using a d(T) primer carrying an engineered restriction site. 4. PCR amplify the coding sequence and any downstream sequences preceding the poly (A) tail using amino-specific and d(T) primers. 5. Analyze PCR amplified products (verify with internal probe if available). Isolate product. 6. Digest with appropriate enzymes, clone and sequence. Step #1: Spiders were collected and identified at Natural Product Sciences, Inc., Salt Lake City, Utah as Agelenopsis aperta. Venom glands were pulled from anesthetized spiders and quickly frozen in liquid nitrogen. RNA was extracted from the venom glands using the protocol of Chomczynski and Sacchi (Analytical Biochemistry 162, 156 (1987)). Polyadenylated messenger RNA (nRNA) was purified using oligo d(T) cellulose (Pharmacia LKB, Sweden) chromatography. Step #2: An oligonucleotide corresponding to residues 1 through 8 of the peptide K amino acid sequence was designed using some Drosophila codon preferences to reduce degeneracy. The cDNA synthesis (antisense) primer was composed of a run of 15 deoxythymidine residues adjacent to a Not I restriction enzyme site. An internal oligonucleotide probe was synthesized to represent amino acid sequence 10 to 16 with all possible preferences. The sense primer and internal primer were synthesized at the University of Utah, Howard Hughes Medical Institute contract facility. The d(T) Not I primer was purchased from Promega (Madison, Wis.). Step #3: cDNA synthesis Messenger RNA was reverse transcribed to cDNA with murine leukemia virus reverse transcriptase (Bethesda Research Laboratories, Maryland) using the manufacturer's protocol. The 20 ul reaction mixture contained the enzyme buffer as supplied in a cDNA synthesis kit (Boehringer Mannheim, Indiana), 50 ng of mRNA, 2 units of RNase H, 30 ng of d(T)Not I primer, 1 mM each deoxynucleoside triphosphates, and 100 u of reverse transcriptase. The reaction mixture was incubated for 1 h at 37° C. and continued for 10 minutes at 42° C. The reaction mixture was ethanol precipitated and resuspended in 20 ul H 2 O. Step #4: Amplification Primer directed enzymatic amplification of DNA with a thermostable DNA polymerase was initially described by Saiki, et. al. (Science, 239:487 (1988)). For our application, 10 ul of the venom gland cDNA was used as template in a polymerase chain reaction containing reagents contained in the GeneAmp™ DNA amplification kit (Perkin Elmer Cetus, California). The amplification reaction contained the sense and antisense primers in a 2 uM concentration, 100 uM of each deoxynucleotide triphosphate, and 4 units of the thermostable recombinant Taq polymerase. The reaction was run in a programmable heat block manufactured by Coy Laboratories (Ann Arbor, Mich.). It was started by denaturing the RNA-cDNA hybrid at 95° C. for 2 minutes, annealing the primers for 2 minutes at 37° C., and then extending the primers at 72° C. for 1 minute. This cycle was repeated twice and the program then switched to an identical profile incorporating an elevated annealing temperature of 54° C. This cycle was repeated 35 times. After the final cycle, the samples were chilled at 5° C. Step #5: One-tenth of the reaction mixture was run on a composite gel containing 3% NuSieve/1% SeaKem agaroses (FMC, Rockland, Me.) in Tris/borate/EDTA (TBE) buffer in the presence of ethidium bromide The gel was photographed and transferred onto Biotrace™ nylon membrane (Gelman Sciences, Ann Arbor, Mich.) using an alkaline transfer protocol (E. M. Southern, J Mol Biol 98:503 (1975) and Reed and Mann, NAR 13:7277 (1985)). The membrane was prehybridized for 12 h at 42° C. in 5X SSC (0.75M NaCl/0.075M sodium citrate pH 7.0,) 20 mM sodium phosphate, pH 6.7, 2X Denhardt's (Sigma), and 0.1 mg/ml E. Coli tRNA (Boehringer Mannheim). To the buffer was then added 2×10 5 cpm/ml of 5' end 32 p labeled internal probe. Hybridization was continued at 42° C. for 24 h, and washing was done at 56° C. with 5X SSC/. The blot was autoradiographed using Kodak XAR film at -70° C. for 1 hour. Step #6: The anchored PCR product visualized in FIG. 2 was purified through a Centricon-100 (Amicon) to remove unincorporated primers. The insert was then digested with the restriction enzyme Not I (MBR, Milwaukee, Wis.), utilizing the restriction site contained in the downstream primer. The vector, pKS (Statagene, LaJolla, Calif.), was double digested with EcoR V (U.S. Biochemical) and Not I to generate sites specific for directional cloning. Vector and insert were ligated and transformed into competent E.coli strain DH5α. Colony lifts were screened with the 32 P labeled internal probe and candidate colonies were further characterized by sequencing (U.S. Biochemical's Sequenase Version 2.0) mini-prep DNA using the internal probe as primer. The inserts of three gene-containing candidates were then sequenced in entirety using commercially available external primers The sequence of one of the clones, pKS-KK, is presented in Table 4. The entire coding sequence as well as the 135 bp region preceding the poly (A) tail account for 282 base pairs of the sequence for the AG1 gene. Analysis of the translated peptide sequence suggested a protein of MW 5,280.73 daltons with 8 cysteine residues as shown in Table 5. If the cysteines are involved in disulfide bridges this suggests a molecular weight of 5,272.73 daltons which is in agreement with the mass spectroscopy data. EXAMPLE 4 The high molecular weight Agelenopsis aperta toxin described in Example 1 was obtained using the same procedure as described in Example 1. Gel-filtration fractions having the effects described in Example 1 were diluted 1:150 with Tyrode and bath-applied for one minute to an in vitro preparation of the chick brain stem. This brief exposure to dilute toxin produced partial (about 50%) blockade of transmission. These partial blocks were stable for at least one hour and were unaffected by increases or decreases in the rate of cochlear nerve stimulation over a range of 0-30 Hz. Partial blockade, however, was largely reversed by increasing extracellular calcium from 3 to 9 mM. Subsequent reduction of extracellular calcium back to 3 mM caused the postsynaptic response to revert to its previous level of partial blockade. The result so obtained indicates that this toxin acts to produce long-lasting blockade of transmission. The finding that the effects of this toxin are independent of stimulation frequency suggests that the toxin does not act primarily on synthesis or reuptake of transmitter. The inverse relationship between extracellular calcium concentration and the blocking effects of the toxin indicates that the toxin likely acts on calcium channels. This action could be exerted on presynaptic calcium channels necessary for the release of transmitter and/or on postsynaptic calcium channels involved in the response of the cochlear nucleus neurons to synaptic stimulation. EXAMPLE 5 The toxin AG1 described in Examples 1 through 4 is obtained in the manner described above from Agelenopsis aperta spider venom. The toxin so obtained is applied and a complete blockade of synaptic transmission is achieved as described in Example 1. EAA agonists quisqualic acid and kainic acid are then individually bath-applied to the cochlear nucleus neurons at concentrations of 5 mM and 50 uM, respectively. Application of these agonists at such concentrations normally reduces the ability of cochlear nucleus neurons to respond to direct electrical stimulation, presumably by depolarizing them by an action on EAA receptors. When applied in the presence of AG1 toxin the same effect is seen; that is, after 10 minutes of application of either quisqualic of kainic acids in the presence of AG1 toxin the response of cochlear nucleus neurons to direct antidromic stimulation is reduced by about 75%. The degree and time-course of this effect are not significantly different from those observed when either quisqualic of kainic acid is applied in the absence of AG1 toxin. The result so obtained indicates that this toxin does not exert its blocking effects on synaptic transmission by a direct action on EAA receptors on the postsynaptic neuron but rather by a direct action of the toxin on calcium channels as suggested in Examples 1 and 3. If it were acting directly on EAA receptors it would be expected that the toxin would also block the effects of directly applied EAA agonists, such as quisqualic or kainic acid. The obtained result runs counter to that expectation. EXAMPLE 6 The high molecular weight toxin described in Examples 1 through 5 (AG1) is obtained in the manner described above from Agelenopsis aperta spider venom. The toxin so obtained is applied for the purpose of labeling calcium channels in neurons or other cell types. A radioactive label (such as 125 I) is incorporated in the toxin molecule. Binding of the labeled toxin is then assayed using autoradiography of tissue sections or quantification (using scintillation counting) of binding to various tissue extracts such as synaptosomal or membrane preparations. Autoradiography of brain tissue sections labeled with the radioactive toxin reveals the regional distribution of the calcium channels to which the toxin binds. (A similar result is obtained by observing the binding pattern of the toxin conjugated to a fluorescent label.) The binding of the toxins to tissue extracts under various conditions, such as the presence of other drugs, provides information regarding the pharmacology of the calcium channel to which the toxin binds. EXAMPLE 7 The AG1 toxin described in Examples 1-6 above is obtained in the manner described above from Agelenopsis aperta spider venom. The toxin so obtained is tested for its ability to block synaptic transmission in an in vitro brain slice preparation of the rat hippocampus following generally the procedures described by Mueller et al. ("Noradrenergic responses in rat hippocampus: Evidence for mediation by alpha and beta receptors in the in vitro slice." Brain Research 214:113-126, 1981). When dissolved in the artificial cerebral spinal fluid bathing the hippocampal slice, the toxin shows a concentration-dependent ability (over the range from 1 nM to 1 μM) to inhibit the population spike (PS) recorded extracellularly from the CA1 region of the hippocampus with microelectrodes. In repeated experiments, the toxin is shown to produce half-maximal inhibition of the PS at a concentration of approximately 100 nanomolar. At concentrations that completely inhibit the PS, this toxin has no effect on axonal conduction, as reflected in the unchanged "afferent volley" component of the measured response. The magnitude of the PS suppression produced by this toxin is reduced as extracellular calcium concentration is increased from 2.4 mM (control) to 7.5 mM and 10 mM. IV. SUMMARY It will be appreciated that the present invention provides the ability to effectively block specific channels using the toxin. Similarly, specific channel blockers with activity on the central nervous system may have the potential to treat various neurological disorders. It has been found, for example, that these channel blockers may act as a treatment of epilepsy. In addition, channel blockers of the type disclosed in the present invention may also be used in treatments of stroke, traumatic head injury, and degenerative central nervous system diseases such as Huntington's disease. In summary, it can be seen that the methods and compositions of the above invention accomplish the objectives set forth above. In particular, the present invention provides calcium channel blockers which can be used as research tools or in a clinical setting. In particular, the spider toxins of the present invention can be used as calcium channel blockers in the central nervous system. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within their scope. | Summary: Methods and compositions for blocking Ca 2+ channels within an organism are provided. For example, a toxin was isolated from the Agelenopsis aperta spider. The toxin comprised a 48 amino acid toxin having a molecular weight of approximately 5,274. This toxin was found to block calcium channels within the central nervous system. The Agelenopsis gene responsible for producing this toxin has been identified and cloned. This gene and/or its derivatives provide a mechanism by which the toxin can be produced using recombinant DNA expression technologies. The present invention further relates to methods of treating neurological diseases by applying the toxins isolated and identified. The toxin may provide beneficial effects on certain neurological conditions including seizures, ischemic-hypoxic CNS damage, and neurodegenerative disorders. It is also found that the toxins are effective as tags in probing calcium channels. | 12,728 | 190 | big_patent | en |
Write a title and summarize: Eine Hauptstadt ist ein symbolisches, zumeist auch politisches Zentrum eines Staates und oft Sitz der obersten Staatsgewalten: Parlament, Monarch, Regierung, Oberstes Gericht. Dieser Status ist oftmals per Verfassungsgesetz deklariert. Selten weicht der Regierungssitz ab; so zum Beispiel in den Niederlanden, Bolivien, Tansania und Malaysia sowie von 1990 bis 1999 in Deutschland. Auch gibt es Hauptstädte, die räumlich vom judikativen Verfassungsorgan getrennt sind, so in Deutschland (Karlsruhe) und Tschechien (Brünn). Vor allem in Bundesstaaten können die obersten Organe der Staatsgewalt auf mehrere Städte verteilt sein. Oft gibt es abweichende Finanz-, Industrie-, Verkehrs-, Wissenschafts- und Kulturzentren. == Hauptstadt als wichtigstes Zentrum Sehr oft entwickelte sich die Hauptstadt über einen langen Zeitraum hinweg zum unangefochtenen Herzstück eines Nationalstaats. Sie ist nicht nur politisches Zentrum (Residenzstadt), sondern auch Mittelpunkt der Industrie, Wissenschaft, Kunst und Kultur einer Region (Hauptort). Im Städtebau einer Hauptstadt machen sich die Hauptstadt- und Regierungsfunktionen häufig durch monumentale und auf staatliche Selbstdarstellung zielende Gebäude, Straßenzüge, Plätze und Grünanlagen sowie durch die Anlage eines Regierungsviertels bemerkbar. In Frankreich konnte sich die Hauptstadt Paris zur bedeutendsten Stadt des Landes entwickeln. Bereits im Mittelalter wurde der Grundstein für diese Entwicklung gelegt, als die französischen Könige die Stadt zu ihrer Hauptstadt machten, indem sie damit begannen, zentralörtliche, oft höfische, später zunehmend staatliche Funktionen an diesem Ort zu konzentrieren (Zentralismus). 1257 entstand etwa mit dem Gymnasium College de Sorbonne der Vorgänger der ältesten und bekanntesten Universität Frankreichs. Franz I. ließ im 16. Jahrhundert Künstler wie Michelangelo, Tizian oder Raffael nach Paris kommen und legte damit den Grundstock für die königliche Gemäldesammlung, die heute der Louvre beherbergt. In den folgenden Jahrhunderten entstanden weitere für Frankreich bedeutende Einrichtungen wie z. B. die Academie francaise. Trotz der Verlagerung der Residenz von Paris nach Versailles unter Ludwig XIV. blieb die Stadt weiterhin das politische und wirtschaftliche Zentrum. Die Stadt wurde weiter ausgebaut, sie beherbergte zwischen 1855 und 1937 sechs Weltausstellungen. Während der Belle Epoque wurde die Stadt erneut zu einem international anerkannten kulturellen und intellektuellen Zentrum. Noch in den letzten Jahrzehnten errichteten französische Präsidenten monumentale Bauwerke wie den Grande Arche, was die Bedeutung der Stadt weiter unterstrich. Paris ist auch heute nicht nur Zentrum der Kultur und Politik, die Stadt ist größter Verkehrsknotenpunkt und größter Wirtschaftsraum in Frankreich. Es fahren auch viele Besucher in die Hauptstadt. Auch London durchlief zunächst für England, später für das ganze Vereinigte Königreich eine vergleichbare Entwicklung. Weitere Beispiele sind Mexiko-Stadt (Mexiko), Buenos Aires (Argentinien) oder Bangkok (Thailand). == Hauptstadt als vorwiegend politisches Zentrum Von der weit verbreiteten Norm, als Hauptstadt auch das kulturelle und wirtschaftliche Zentrum zu sein, weichen viele Hauptstädte aus verschiedenen historischen oder politischen Gründen ab. So ist die Hauptstadt nicht immer gleichzeitig die größte und bedeutendste Stadt. Ihre Bedeutung beschränkt sich oftmals nur auf die Funktion als Regierungssitz. Oftmals wandeln sich allerdings im Laufe der Jahre anfänglich bedeutungslose, zu Hauptstädten ernannte Städte durch ihren erlangten Status zu wichtigen Zentren über ihre administrative Bedeutung hinaus. Die Hauptstadt der Vereinigten Staaten, Washington, D.C., belegt in der Reihung nach Einwohnerzahlen nur Platz 27 (auf Rang 1 steht New York City). Aufgrund der geografisch zentraleren Lage und als Zeichen der Abgrenzung vom Osmanischen Reich wurde die Hauptstadt der neu gegründeten Türkischen Republik 1923 von der Metropole Istanbul ins deutlich kleinere und vergleichsweise unbedeutende Ankara verlegt. Die zuvor eher unbedeutende kanadische Stadt Ottawa wurde aufgrund ihrer Lage an der englisch-französischen Sprachgrenze und somit besseren Akzeptanz für beide Bevölkerungsteile als Hauptstadt ausgewählt. Man ging für eine Kleinstadt auch von einer geringeren Bedrohungslage im Kriegsfall aus. Kasachstan verlegte 1997 seine Hauptstadt von Almaty in das halb so große Astana (heute Nur-Sultan). Zum einen geschah dies wegen der Erdbebengefahr in Almaty, zum anderen wählte man die Stadt in der ungefähren Mitte des Landes zwecks besserer Kontrolle der russischsprachigen Minderheit im Norden. Im Jahr 1949 wurde in der Bundesrepublik Deutschland die bis dahin wenig bedeutende Stadt Bonn zur (provisorischen) Bundeshauptstadt gewählt. Dies geschah wohl vor allem auf Initiative des Rheinländers und ersten Bundeskanzlers Konrad Adenauer. Ein weiterer Grund für den Erfolg Bonns gegenüber dem Mitbewerber Frankfurt am Main war vermutlich die Befürchtung, dass sich die Bevölkerung nach einer Wiedervereinigung für die Beibehaltung Frankfurts als Hauptstadt hätte aussprechen können. Bei Bonn handelte es sich bei Weitem nicht um die größte Stadt der alten Bundesrepublik; Bonn hatte zu dieser Zeit etwa 115.000 Einwohner, Hamburg z. B. 1,6 Millionen. Im Laufe der Jahre wurde Bonn für die Aufgaben als Hauptstadt ausgebaut; die anfänglich hämisch als Bundesdorf, Bundeshauptdorf oder Konradopolis in Anspielung auf Adenauer bezeichnete Stadt konnte durch Eingemeindungen ihre Einwohnerzahl mehr als verdoppeln. Mit der Wiedervereinigung am 3. Oktober 1990 wurde Bonn durch Berlin als gesamtdeutsche Hauptstadt (wieder) abgelöst. Im Berlin/Bonn-Gesetz wurde Bonn (nun mit dem Zusatztitel "Bundesstadt") jedoch der Verbleib zahlreicher Bundesministerien und die Zuordnung mehrerer vormals in Berlin ansässiger Bundesbehörden zugestanden, so dass Berlin keine zentralistische Hauptstadt ist. Für das untergegangene Preußen kann man Berlin jedoch durchaus eine mit London oder Paris vergleichbare Entwicklung und Funktion attestieren. Bern ist de facto Hauptstadt der Schweiz, gilt aber offiziell als Bundesstadt. Siehe dazu Hauptstadtfrage der Schweiz. == Geplante Hauptstadt Die Gründe für den Entschluss eines Staates, eine Planhauptstadt zu errichten, sind vielfältig. Pakistan entschloss sich für den Bau der neuen Hauptstadt Islamabad, da man Vorbehalte gegenüber der Konzentration von Investitionen in der bisherigen Hauptstadt Karatschi hatte. Der Grund für den Bau der brasilianischen Hauptstadt Brasilia lag darin, dass man Bedarf nach einer neutralen und föderalen Hauptstadt hatte. Zudem sollte die nun im geographischen Zentrum liegende Stadt die Entwicklung des Binnenlandes fördern, was mit der an der Küste liegenden vorherigen Hauptstadt Rio de Janeiro so nicht möglich gewesen wäre. In den letzten hundert Jahren entstanden weltweit eine Reihe künstlicher Hauptstädte. Die bekanntesten sind neben den schon genannten wohl Abuja in Nigeria, Canberra in Australien, Neu-Delhi in Indien und 2005 Naypyidaw in Myanmar. Schon in vergangenen Jahrhunderten errichteten Herrscher und Staatsführungen auf dem Reißbrett entstandene Hauptstädte. 1703 ließ Zar Peter I. in den Sümpfen der Newa-Mündung den Grundstein für die neue russische Hauptstadt Sankt Petersburg legen. Von 1712 bis 1918 löste sie Moskau als Hauptstadt ab. Auch Washington, D.C. ist eine geplante Hauptstadt. Gegen Ende desselben Jahrhunderts wurde 1792 an den Ufern des Potomac River mit dem Bau der Hauptstadt der USA begonnen. Am 11. Juni 1800 wurde Washington offiziell zur Hauptstadt. Auch in Deutschland ließen absolutistische Herrscher des 17. und 18. Jahrhunderts Residenzstädte völlig neu entstehen. Ein Beispiel dafür ist die ehemalige badische Landeshauptstadt Karlsruhe. Markgraf Karl Wilhelm von Baden-Durlach ließ am 17. Juni 1715 den Grundstein für die nach ihm benannte Stadt legen. Die strahlenförmige Anordnung der Straßen und Alleen, in deren Zentrum das Residenzschloss liegt, ist heute noch gut zu erkennen, ihr verdankt die Stadt den Beinamen "Fächerstadt". == Von der Hauptstadt abweichender Regierungssitz Der Regierungssitz einiger weniger Staaten befindet sich nicht in der Hauptstadt. So ist Amsterdam sowohl die größte Stadt der Niederlande als auch deren nominelle Hauptstadt, offizieller Regierungssitz und königliche Residenz ist jedoch Den Haag. In Südafrika verteilt sich der Sitz der Verfassungsorgane sogar auf drei Städte, wobei die größte Stadt (Johannesburg) nicht dazugehört. Das Parlament tagt in Kapstadt, das Verwaltungs- und Regierungszentrum ist Pretoria (Tshwane) und die obersten judikativen Einrichtungen (Gerichtshöfe) befinden sich in Bloemfontein. == Staaten ohne bzw. mit nicht international anerkannter Hauptstadt De jure keine Hauptstadt haben Monaco, Nauru, die Schweiz und die Vatikanstadt: In Liechtenstein gibt es keine Stadt. Vaduz wird daher als Hauptort des Fürstentums bezeichnet. == Hauptstadt von Teilstaaten Auch Teilstaaten (beispielsweise Länder in Deutschland und Österreich oder Bundesstaaten in den USA) haben Landeshauptstädte, die für ihren Landesteil außer den politischen auch die übrigen Hauptstadtfunktionen aufweisen. Im Land Preußen gab es auch Provinzhauptstädte. Jeder Schweizer Kanton kennt einen Hauptort, mit Ausnahme des Kantons Appenzell Ausserrhoden, das de jure keinen Hauptort kennt (de facto ist es Herisau). == Hauptstädte von Staaten Dabei handelt es sich um eine teils historische, teils aktuelle Aufstellung der Hauptstädte der einzelnen Staaten, ihrer eventuellen Vorgängerstaaten (z. B. Deutscher Bund, Deutsches Reich), der obersten Verwaltungseinheiten (Bundesstaaten, Länder, Bundesländer, Provinzen) und der abhängigen Gebiete. == Staaten und subnationale Entitäten mit mehreren Hauptstädten == Kleinste und größte Hauptstädte der Welt Angaben basieren auf Volkszählungen gemäß Liste der Hauptstädte der Erde. Berechnungen auf Grundlage von World Gazetteer. == Hauptstädte, die nicht größte Städte ihres Landes sind Insgesamt gibt es derzeit 39 Staaten auf der Welt, deren Hauptstädte ihrer Einwohnerzahl nach nicht an erster Stelle des Landes stehen. Das sind wie folgt: | Title: Hauptstadt Summary: Eine Hauptstadt ist oft die wichtigste Stadt eines Staates. Gemeint ist: In dieser Stadt befinden sich die Organe des Staates. Das sind das Parlament, also die Vertretung des Volkes, ausserdem die Regierung und andere Organe wie das wichtigste Gericht. Auch Landesteile wie die Bundesländer in Deutschland und Österreich oder die Kantone in der Schweiz haben Hauptstädte. Hauptstadt ist eine Stadt also vor allem wegen der Politik. Oft sieht man dort auch viele wichtige Universitäten und Unternehmen. Radio und Fernsehen werden dort gemacht. Man nennt die Stadt ein "Zentrum" für Kultur und Wirtschaft. Manchmal hat der Staat ausdrücklich gesagt, dass eine bestimmte Stadt die Hauptstadt sei. Es kann aber auch sein, dass die Einwohner von sich aus das so empfinden. == Hat ein Land immer nur eine Hauptstadt? Wien ist die Hauptstadt von Österreich. Sie ist ein gutes Beispiel für eine Stadt, die wirklich die wichtigste Stadt im Staat ist. In Wien leben etwa ein Siebtel aller Österreicher. Sie ist viel grösser als alle anderen Städte in Österreich. Das Parlament und alle anderen Organe des Bundesstaates sind dort. Wer was auf sich hält, lebt wahrscheinlich in der Hauptstadt. In anderen Ländern sieht das anders aus. In den Niederlanden zum Beispiel ist die Hauptstadt Amsterdam. Dort hat auch der König seinen wichtigsten Palast, das Paleis op den Dam. Die Regierung und das Parlament arbeiten allerdings in Den Haag. In Südafrika gibt es drei Hauptstädte: Die Regierung arbeitet in Pretoria, in Kapstadt ist das Parlament, und in Bloemfontein steht das oberste Gericht. In Deutschland ist Berlin die Hauptstadt. Das steht sogar im Grundgesetz, dem wichtigsten Gesetz Deutschlands. Berlin ist die grösste deutsche Stadt, und dort befinden sich das Parlament und viele Gebäude der Regierung. Allerdings stehen Gebäude der Regierung auch in anderen Städten, vor allem in Bonn. Zwei der wichtigsten Gerichte sind in Karlsruhe. == Bleibt eine Stadt für immer Hauptstadt? Manchmal bekommt ein Land eine neue Hauptstadt. Wenn die alte schon sehr gross ist, hat man oft keinen Platz für das "Regierungsviertel". Dort sind die wichtigsten Gebäude des Staates. In einer ganz neu gebauten Stadt plant man so ein Viertel von vorneherein ein. Ausserdem empfindet man so eine neue Stadt mehr als die Hauptstadt des ganzen Landes. Ein Beispiel dafür kennt man aus den USA. Zunächst war die Hauptstadt Philadelphia, eine der älteren Städte des Landes. Einige Jahre später wollte man aber eine neue Hauptstadt. Das wurde Washington, benannt nach dem ersten Präsidenten George Washington. Sie lag damals an der Grenze zwischen den nördlichen und den südlichen Teilen der USA. Gerade in Asien, Afrika und Südamerika gibt es viele Länder, die eine neue Hauptstadt bekommen haben. In Afrika liegt zum Beispiel das Land Nigeria. Die alte Hauptstadt an der Küste heisst Lagos. Sie gibt es schon seit dem Mittelalter und hat über 10 Millionen Einwohner. Seit dem Jahr 1991 ist aber Abuja Hauptstadt, das in der Mitte des Landes liegt. Dort wohnen mittlerweile aber auch schon anderthalb Millionen Menschen. | 2,167 | 628 | klexikon_de | de |
Summarize: The body of Sydney Loofe, 24, was found, according to her family. She’d been missing for more than two weeks after going on a date with a woman she met online. Lincoln Police Department Courtesy photo CLOSE “Ready for my date,” were the last four words anyone heard or read from Sydney Loofe on November 15th. Three weeks later, her body was found. Buzz60 Sydney Loofe was last seen on the evening of November 16. (Photo: FBI via Twitter) Authorities have recovered the body of a Nebraska woman whose disappearance after a Tinder date last month triggered a massive search and bizarre social media posts from two persons of interest in the tragic mystery. Lincoln Police Chief Jeff Bliemeister said Tuesday that "analysis of digital evidence" led authorities to a body in rural Clay County they believe is that of Sydney Loofe, 24, who vanished three weeks ago. "We do believe that there is evidence of foul play," Bliemeister said. Bliemeister expressed a "strong belief" that the body is that of Loofe, who was reported missing Nov. 16 after failing to show up at her job at a Lincoln home improvement store. He said formal confirmation would be made in the coming days. Bliemeister provided no further details on the cause of death or circumstances surrounding the discovery. Investigators had been using Loofe's cellphone signal to retrace her movements in the hours before she disappeared. More: Police: Missing Florida teen found safe in New York More: Hunter charged in killing of neighbor while shooting after sunset Loofe’s parents, George and Susie Loofe, acknowledged their daughter's death on their "Finding Sydney Loofe" Facebook page. "It's with heavy hearts that we share this most recent update with you all," the couple said. "Please continue to pray for Sydney and our entire family. May God grant eternal rest unto thee. We love you Sydney." Bliemeister said the persons of interest, Aubrey Trail, 51, and Bailey Boswell, 23, remained in custody but had not been charged in the case. Both apparently left the state in the days after Loofe disappeared and were arrested Thursday near Branson, Mo., on unrelated charges. Social media posts indicate Loofe went on a date Nov. 15 with Boswell, who has confirmed on social media that she met Loofe via the dating app Tinder. Boswell and roommate, Aubrey Trail, 51, live in the eastern Nebraska town of Wilber, about 40 miles south of Lincoln and the last place Loofe was seen alive. Trail and Boswell posted videos on social media last week proclaiming their innocence and claiming their efforts to speak with Lincoln police had been largely rebuffed. Boswell, wearing a hoodie and sunglasses in a video, said she dropped Loofe off at a friend’s house after their date and never heard from her again. Bliemeister said authorities thus far have been unable to confirm Boswell's timeline. Trail said on his video that he "wasn't running from anything." He said he was praying for Sydney and wished the best for her family. "We're continuing to speak with Aubrey Trail and we'll continue to do so as long as he's willing," Bliemeister said Tuesday. Bliemeister said police believe that there is no continuing threat to the public. But he provided no motive for the murder and stressed that no one had been charged. "By their own statements on social media, we believe that Aubrey Trail and Bailey Boswell were two of the last people to see her before her disappearance," Bliemeister said. "Thus they remain persons of interest." Read or Share this story: https://usat.ly/2nvPwxp The interactive transcript could not be loaded. Rating is available when the video has been rented. This feature is not available right now. Please try again later. | Summary: Sydney Loofe sent friends a selfie on Snapchat on Nov. 15 with the caption, "Ready for my date." It was the last time friends would hear from Loofe, who went on a date with a woman she'd met on Tinder but didn't turn up for work the next day, police say, per the Washington Post. On Tuesday, their three-week search for the 24-year-old Nebraska woman came to a close with the discovery of a body in Clay County, about 90 miles from Loofe's Lincoln Home, per the Kansas City Star. Foul play is suspected, Lincoln Police Chief Jeff Bliemeister tells USA Today. Bailey Boswell, 23, who posted a video online in which she said she dropped Loofe off at a friend's house after their date, and her male roommate, 51-year-old Aubrey Trail, are considered persons of interest in the case, police add. Both were jailed last week on unrelated charges. Police haven't commented on how Loofe is believed to have died, or on a possible motive for her killing, but they say "digital evidence" led them to the body. They also say data from Loofe's cellphone indicated she had been in the area of Boswell and Trail's Wilber home, 40 miles from her own. But though Boswell says Loofe smoked marijuana there on Nov. 15, she denied any wrongdoing in a nine-minute video posted online on Nov. 29, showing Boswell and Trail together in a car, per the Omaha World-Herald. A day later, Boswell and Trail were arrested near Branson, Mo. "By their own statements on social media, we believe that Aubrey Trail and Bailey Boswell were two of the last people to see [Loofe] before her disappearance," Bliemeister says. "Thus they remain persons of interest." | 853 | 416 | multi_news | en |
Summarize: A disgraced town crier has been stripped of his post after falsely boasting that he had a distinguished military career and wearing war medals he bought online. Anthony Church was often seen around Oxfordshire wearing the replica medals, having allegedly claimed he was a regimental sergeant major with the Coldstream Guards during the Falklands War. The 62-year-old, from Cowley, Oxford, wore the Order of the British Empire, the South Atlantic Medal for those who fought in the Falklands, and a General Service Medal with a Northern Ireland clasp. Mr Church, who worked as a town crier in Banbury, Oxford, Thame, Chipping Norton and Wallingford, today apologised for his 'grave error in judgment' Town crier Anthony Church (pictured left and his medals, right), from Cowley, Oxford, has been stripped of his post after falsely claiming he had a distinguished military career. The 62-year-old (pictured with fellow town criers from around the country) often wore the British Empire Medal and the South Atlantic Medal for those who fought in the Falklands which he had purchased online. He also used the title BEM - which stands for British Empire Medal - after his name on Banbury Town Council's website and apparently wore the award as well. But Mr Church was today forced to apologise for his 'grave error of judgement' after it emerged that he had never served in the Armed Forces. Instead, Mr Church admitted he had bought the medals off the internet before wearing them in public, including at Remembrance Day parades. He has now been forced to step down from the Loyal Company of Town Criers and the Ancient and Honorable Guild of Town Criers. Issuing an apology, Mr Church, who worked as a town crier in Banbury, Oxford, Thame, Chipping Norton and Wallingford, said he had'made a mistake'. He claimed the BEM medal was awarded to his late father Jack Church for taking part in the Berlin Airlift and that he mistakenly believed he was entitled to wear it. He added that he wore the other medals as a 'tribute' to former servicemen. He said: 'I was told several years ago that as the sole-surviving son I was entitled to wear the BEM and put BEM after my name. 'Tribute': The disgraced town crier claims the medals were worn to honour the Armed Forces. Church used the title BEM after his name, which stands for British Empire Medal, pictured, and apparently wore the award as well. 'I also wanted, with the anniversary of the Falklands and World War One, to show my solidarity for those people who had served in these campaigns and found a place I could purchase replica medals and purchased a South Atlantic Medal. 'In September I contacted Buckingham Palace on an unrelated matter and received a letter back asking when I had been awarded the BEM. 'I wrote back explaining the situation and was told that I had been misinformed and was in fact not allowed to use the title or wear the medal as it would lead people to assume that I had been awarded the medal, so I immediately removed all medals.' He added: 'I realise now that I made a grave error of judgement with this. 'It was very stupid and I categorically now apologise to everybody who has served in the forces. It was never my intention to cause any distress or upset. 'I made a mistake, I told someone I'd served and it's been going on from there. I can't apologise enough for the hurt and distress it has probably caused people. 'People will probably feel, with hindsight, that I have misled them. I was in the wrong. I did not mean to offend anyone in the military - it was meant to be a show of support. Mr Church also used the title BEM - which stands for British Empire Medal - after his name on Banbury Town Council's website. 'It was never my intention to cause any distress but it has backfired and cost me everything.' Mr Church was exposed by a group calling itself the Walter Mitty Hunters Club, a group of former servicemen which investigates those who illegitimately wear medals. The Royal British Legion said medals awarded to a deceased service or ex-service people may be worn on the right breast by a near relative. Mr Church wore the medals he acquired on the left. On their Facebook page, John Theman, Secretary of The Loyal Company of Town Criers, said: 'We wish to thank The Walter Mitty Hunters Club for outing Anthony Church. 'We wish to make it known that his actions are deplorable and beneath contempt. 'He has resigned from all Town Crier positions, but rest assured, even if he hadn't resigned, he would have been thrown out of our organisation in disgrace. 'A month ago we investigated his wearing of medals, and he apologised profusely, saying it was a one-off, and we accepted his apology. However the fact he claimed the awards of MBE and BEM really is astounding. 'The Loyal Company of Town Criers has immediately removed all trace of Anthony Church from our websites. 'To the many genuine Town Criers in the UK and beyond, this will come as a huge shock, so, once again, well done sirs.' Chairman of the Oxfordshire Royal British Legion, Jim Lewendon said: 'I saw Mr Church wearing the medals at Remembrance time on his left breast and assumed they were his. 'And a few years back he told me he had been in the Coldstream Guards. Wearing the medals like this is an insult to the bravery of the troops who served. 'I can't believe Anthony was a pretender and I hope he can put this behind him.' In 2012 Mr Church, from Cowley, in Oxford, led Team GB athletes to the spectacular opening ceremony for the London Olympics. He was one of three representatives of the Ancient & Honourable Guild of Town Criers who dressed in traditional liveries to help marshall athletes from 205 nations from the Olympic Village to the stadium in time for the Athletes' Parade | Summary: Anthony Church, 62, wore several medals, including British Empire Medal. He allegedly claimed to have served in Falklands with Coldstream Guards. Mr Church apologised for 'grave error' saying medals were worn in tribute. He claimed he mistakenly thought he could wear his father Jack's BEM. Loyal Company of Town Criers said Mr Church's actions were 'deplorable' Royal British Legion spokesman said it was an 'insult to bravery of troops' Mr Church was town crier for Banbury, Oxford, Thame, Chipping Norton and Wallingford. | 1,380 | 135 | cnn_dailymail | en |
Summarize: Background This section describes DOE’s requirements and guidance for AOA; DOE and NNSA offices responsible for conducting AOAs for capital asset projects; and NNSA’s recent projects that have completed, or are nearing completion of, an AOA. DOE’s Requirements and Guidance for Analysis of Alternatives DOE’s mandatory requirements and optional guidance for identifying, analyzing, and selecting alternatives when conducting AOAs also apply to NNSA. DOE’s requirements for the AOA process are outlined in Order 413.3B governing the Program and Project Management for the Acquisition of Capital Assets (hereafter referred to as Order 413.3B). This order includes requirements for the acquisition of capital assets, with the stated goal of delivering fully capable projects within the planned cost, schedule, and performance baseline. In addition to the order’s requirements, DOE has guidance for identifying, analyzing, and selecting alternatives (hereafter referred to as “guidance”) that is found throughout seven guides associated with the order. DOE states at the beginning of each of these guides that they include nonmandatory approaches for meeting requirements, that guides are not requirements documents, and that they are not to be construed as requirements in any audit for appraisal of compliance with the parent policy or order. DOE’s Order 413.3B establishes five critical decision processes of project development that each end with a major approval milestone—or “critical decision point”; these decision processes cover the life of a project. Under Order 413.3B, an AOA occurs during the span of the first two critical decision processes—the preconceptual design process (CD-0) and the conceptual design process (CD-1)—with the majority of the AOA being conducted during the conceptual design process and ending with CD-1 approval. Figure 1 illustrates when DOE conducts the analysis of alternatives as part of its project management process. DOE’s Order 413.3B and its associated guides include the AOA-related requirements and guidance listed below, among others. Appendix II includes a full description of DOE’s requirements and guidance for conducting an AOA. As part of the preconceptual design (CD-0) approval process, the mission need—which DOE defines in Order 413.3B as a credible gap between current capabilities and those required to meet the goals articulated in the strategic plan—and functional requirements—the general parameters that the selected alternative must have to address the mission need—must be identified. The mission need must be independent of a particular alternative, and the program office responsible for the capital asset project must explore a variety of alternatives. As part of the conceptual design (CD-1) approval process, reliable cost and schedule range estimates for the alternatives considered must be developed, and whatever figure or range is provided at the CD-0 and CD-1 stages must explicitly note relevant caveats concerning risks and uncertainties inherent in early estimates. In addition, a conceptual design report must be developed that includes, among other things, a clear and concise description of the alternatives analyzed, the basis for the selected alternative, how the selected alternative meets the mission need, the functional requirements that define the alternative and demonstrate that the alternative can be successful, and life-cycle cost assumptions. During the CD-0 and CD-1 approval process, several independent reviews must be conducted, depending on the estimated cost of the project, related to two aspects of the AOA process: validation of (1) the mission need statement and (2) the cost estimates. As a project moves toward approval of CD-2, if the top end of the approved CD-1 cost range for the selected alternative grows by more than 50 percent, the program office must reassess alternatives by conducting another AOA and obtaining another CD-1 approval. DOE’s guides suggest summarizing a planned approach to conduct an analysis of alternatives; considering at least three viable alternatives for analysis, including one that represents the status quo; developing cost estimates that are explicit ranges instead of point estimates; including life-cycle cost estimates of the alternatives being considered; quantifying the benefits of alternatives over their life cycle; adjusting life-cycle cost and benefit estimates for risk; considering various selection criteria for the alternatives; weighting the selection criteria for relative importance; and comparing alternatives using net present value.The guidance suggests that, at the end of the analysis, the AOA team present the recommended alternative based on the preceding analysis in an integrated form, summarizing why an alternative is preferred and supporting the recommendation of the preferred alternative with facts from the analysis. In addition, DOE’s guidance suggests that the program office review the alternative selection and cost range to assess whether the AOA process (1) evaluates a range of appropriate attributes for each alternative, including cost, risks, safety, technology and regulatory requirements, and (2) is reasonable and provides best value to the government. Order 413.3B also requires prior to CD-1 approval that DOE conduct a separate analysis of alternatives under the National Environmental Policy Act of 1969 (NEPA). Under NEPA, federal agencies must identify and assess the likely environmental effects of proposed projects, and reasonable alternatives, using an environmental assessment or, if the projects likely would significantly affect the quality of the human environment, a more detailed environmental impact statement. Recognizing the need for better integration between the AOA conducted under Order 413.3B and the AOA conducted under NEPA, in early 2012, DOE established a multidisciplinary team of NEPA-compliance and program- and project-management specialists, and in June 2012, the Secretary of Energy issued a memo highlighting the importance of integrating these two AOAs. DOE officials who were part of this team stated that DOE has not yet decided how to connect the NEPA process with the AOA process under Order 413.3B. DOE and NNSA Offices Responsible for Conducting AOAs for Capital Asset Projects Within DOE and NNSA, several groups are responsible for various aspects of conducting an AOA: DOE Office of Acquisition and Project Management (DOE APM). This office is responsible for, according to DOE APM officials, writing DOE’s project management Order 413.3B and its associated guides, including requirements and guidance for conducting an AOA, and for reviewing some DOE projects. NNSA Office of Acquisition and Project Management (NNSA APM). This office is responsible for managing construction of capital asset projects within approved cost and schedule estimates, and for conducting at least yearly reviews of the construction projects to evaluate technical, cost, scope, and other aspects of the projects. According to NNSA APM’s Concept of Operations, this office leads the development of the analysis of alternatives for NNSA projects. NNSA program offices. Program offices are responsible for the projects that fall within their portfolios. For projects, according to NNSA APM’s Concept of Operations, the program offices lead the development of the mission needs and functional requirements, support the analysis of alternatives, and lead the selection of the preferred alternative. NNSA Office of Cost Estimating and Program Evaluation. The National Defense Authorization Act for Fiscal Year 2014 mandated the permanent establishment of this office to provide an independent review of cost estimates and to advise the NNSA Administrator on AOA policies and procedures, among other responsibilities. NNSA officials stated that they expected this office to work with other NNSA offices to define its roles and responsibilities as they relate to conducting AOAs during fiscal year 2015. NNSA’s Recent Projects That Have Completed, or Are Nearing Completion of, an AOA The following describes the three recent NNSA projects that we reviewed that had completed, or are nearing completion of an AOA. High Explosive Science, Technology and Engineering Project This project is intended to replace and upgrade existing facilities used for NNSA’s high explosive manufacturing support, surveillance, testing, and technology development program. NNSA began conducting the AOA in November 2011 and expects to complete the AOA process by obtaining CD-1 approval in December 2014. As of September 2014, NNSA estimated that the total project cost would range from $100 million to $155 million at an 85 percent confidence level. Radioactive Liquid Waste Treatment Facility This project is intended to replace an existing facility at the Los Alamos National Laboratory to treat and dispose of two types of radioactive waste. According to this project’s mission need statement document, the existing facility was nearing its designed life span and had suffered numerous failures. NNSA completed an initial AOA in 2006, selecting the alternative to place the treatment capabilities for both types of waste in the same structure. NNSA estimated in 2006 that the total project cost would likely range between $82 million and $104 million. After significant cost increases were incurred during the preliminary design process of the project, NNSA completed a reassessment of alternatives in September 2013, which we examine in this report. NNSA indicated in this reassessment that the existing facility was beyond its designed life span. The selected alternative for this reassessment separates treatment capabilities into two separate structures, and NNSA estimated that the revised total project cost would likely range between $168 million and $220 million. Uranium Processing Facility This project is intended to replace existing facilities for enriched uranium capabilities that, according to this project’s mission need statement document, require intensive maintenance and are experiencing escalating operating costs. NNSA completed an initial AOA in 2007, selecting the alternative to construct a new building with full capability to process enriched uranium. NNSA estimated in 2007 that the total project cost would likely range between $1.4 billion and $3.5 billion. During the preliminary design phase, the estimated cost of the project increased significantly, and NNSA completed a reassessment of alternatives in June 2012. This second AOA, which we examine in this report, selected a new alternative to construct the same building, but included fewer capabilities, while deferring significant portions of the project’s original scope to be added in the facility at a later time. The approved estimated cost increased to a range of $4.2 billion to $6.5 billion. This independent cost estimate was requested under the National Defense Authorization Act of 2013. The $9.8 billion is a point estimate and not the full cost range. The top end of the Department of Defense’s Office of Cost Assessment and Program Evaluation cost range estimate was at least $10.5 billion with the potential of higher cost depending on constrained or unconstrained funding profile. NNSA and DOE senior management a new alternative of building two new, smaller facilities to include all capabilities approved in the 2012 AOA, and senior management approved this new alternative. NNSA began to consider alternatives other than building a single facility because, according to the UPF Federal Project Director, the project was facing budget constraints, rising costs, and competition from other high- priority projects within NNSA. In January 2014, the NNSA Administrator asked for a peer review to develop and recommend an alternative approach to UPF. In April 2014, the peer reviewers recommended an alternative similar to the one approved by DOE and NNSA senior management in December 2013. This alternative would require NNSA to construct two new, smaller facilities and upgrade existing facilities to cover the same capabilities approved in the 2012 AOA, all within the $6.5 billion approved top end of the cost range. According to NNSA officials, this peer review was not an AOA. According to the NNSA Deputy Federal Project Director for this project, the Federal Project Director provided direction in April 2014 to proceed with the alternative proposed by the peer review. According to NNSA officials in October 2014, the alternative was further modified. At this time, NNSA’s plan was to build three new, smaller facilities and upgrade existing facilities to provide the same capabilities approved in 2012 and still remain within the approved cost range of $4.2 billion to $6.5 billion. GAO Identified 24 Best Practices for the Analysis of Alternatives Process Because no one single set of practices existed that was broadly recognized by government and private-sector entities for the AOA process, we identified 24 best practices for identifying, analyzing, and selecting alternatives. We identified these best practices by (1) compiling and reviewing commonly mentioned AOA policies and guidance used by different government and private-sector entities and (2) incorporating experts’ comments on our draft set of practices to develop a final set of practices. These practices can be applied to a wide range of activities in which an alternative must be selected from a set of possible options, as well as to a broad range of capability areas, projects, and programs. These practices can provide a framework to help ensure that entities consistently and reliably select the project alternatives that best meet mission needs. We grouped these 24 best practices into four categories: (1) general principles, (2) identifying alternatives, (3) analyzing alternatives, and (4) selecting a preferred alternative. The four categories of best practices address the AOA process from defining the mission need and functional requirements to independently reviewing its results. On the basis of our reviews and experts’ comments, we believe that these best practices can be generally applied from the beginning of the AOA process with practices from the general principles category, through practices in the identifying and analyzing alternatives categories, and ending with practices in the selecting a preferred alternative category. We also believe that these best practices do not necessarily have to be followed in order and that some of them can be applied concurrently with other best practices. For example, the best practice of defining the selection criteria based on the mission need in the selecting a preferred alternative category could be addressed at the same time as the best practice of creating a study plan in the general principles category. The following sections describe the 24 best practices by category. General Principles Category The general principles category contains best practices that would need to be applied before starting the process of identifying, analyzing, and selecting alternatives, such as determining the mission need and functional requirements, developing the study time frame and creating a study plan, and determining who conducts the analysis. It also includes best practices that would need to be applied throughout the AOA process, such as documenting all steps taken to identify, analyze, and select alternatives in a single document. Table 1 lists the best practices in the general principles category. Identifying Alternatives Category The identifying alternatives category contains best practices that help ensure the alternatives to be analyzed are sufficient, diverse, and viable. Table 2 lists the best practices in the identifying alternatives category. Analyzing Alternatives Category The analyzing alternatives category contains best practices that are necessary to compare the alternatives selected for analysis. The best practices in this category help ensure that the team conducting the analysis uses a standard, quantitative process to assess the alternatives. This category includes best practices related to estimating the costs and benefits of each alternative over its life cycle and understanding the impacts of risks and key assumptions on these estimates. Table 3 lists the best practices in the analyzing alternatives category. Selecting a Preferred Alternative Category The category for selecting a preferred alternative contains best practices that help ensure the team selects a preferred alternative that best meets the mission needs. This category includes best practices related to defining selection criteria, differentiating among the selection criteria, and independently reviewing the AOA process. Table 4 lists the best practices in this category. On the basis of our reviews and experts’ comments, we believe that conforming to these best practices helps ensure that the preferred alternative that is selected is the one that best meets the agency’s mission needs. Not conforming to best practices may lead to an unreliable AOA, and the agency will not have assurance that the preferred alternative best meets its mission needs. Appendix III lists the best practices from all four categories. DOE’s AOA Requirements and Guidance Do Not Conform to Best Practices Neither DOE’s AOA requirements nor its guidance conform to best practices and, therefore, DOE does not have assurance that applying these requirements and guidance may lead to reliable AOAs. Our review of DOE’s requirements contained within Order 413.3B found that they minimally meet best practices overall, and that they do not fully or substantially meet best practices in any of the four categories. Our analysis also found that, even when DOE’s requirements are combined with guidance contained in the guides associated with Order 413.3B, they only partially meet best practices overall, and they only fully or substantially meet best practices in the identifying alternatives and selecting a preferred alternative categories but not in the general principles or analyzing alternatives categories. DOE’s Order 413.3B Requirements Do Not Conform to Best Practices DOE’s Order 413.3B requirements for the AOA process minimally meet best practices. Therefore, DOE does not have assurance that applying these requirements may lead to reliable AOAs. DOE’s requirements do not fully or substantially meet best practices in any of the four AOA categories, and they conform to only 1 of the 24 best practices we identified—the practice of having the customer define functional requirements based on the mission need. Our assessment of DOE’s Order 413.3B requirements found the following: General principles. DOE’s Order 413.3B requirements minimally meet best practices for the general principles category by substantially meeting 1 of the 8 best practices in this category, partially or minimally meeting 4, and not meeting the other 3. DOE requires that the program office define a project’s mission need without a predetermined solution and that the Integrated Project Team must represent a diverse range of disciplines, develop functional requirements to satisfy the mission need, and document some of the steps and assumptions required as part of conducting the AOA. DOE’s Order 413.3B does not make any reference to appropriate AOA time frames, an AOA study plan, or conducting the analysis without a predetermined solution. Identifying alternatives. DOE’s Order 413.3B requirements minimally meet best practices for identifying alternatives by partially or minimally meeting 2 of the 4 best practices in this category and not meeting the other 2. DOE requires that the project’s customer, not the AOA team, explore a variety of alternatives and that the alternatives be defined clearly and concisely. DOE’s Order 413.3B does not make any reference to including a status quo alternative in the AOA or pre- screening the list of identified alternatives for viability. Analyzing alternatives. DOE’s Order 413.3B requirements minimally meet best practices for analyzing alternatives by partially or minimally meeting 3 of the 8 best practices in this category and not meeting the other 5. DOE requires that life-cycle cost assumptions be included in the conceptual design report, which leaves room for interpretation because it could be interpreted as requiring the inclusion of life-cycle cost estimates for all alternatives or, as DOE and NNSA officials said that they interpret this statement, as requiring a life-cycle cost estimate for only the selected alternative after completion of the AOA process. DOE also requires that project risks and mitigation strategies are assessed. DOE’s Order 413.3B does not make any reference to presenting life-cycle cost estimates in present value terms, quantifying benefits, explaining how measures of benefit support the mission need, or testing the sensitivity of cost and benefit estimates to risks and changes in key assumptions. Selecting a preferred alternative. DOE’s Order 413.3B requirements do not meet best practices for selecting a preferred alternative by minimally meeting 1 of the 4 best practices in this category and not meeting the other 3. The best practice that DOE minimally meets is having an entity independent of the AOA process reviewing the extent to which all best practices have been followed. Order 413.3B contains four separate reviews that might be required during CD-0 and CD-1 processes depending on the amount of the project’s cost estimate, but these reviews are limited to two aspects of the AOA process: the validation of (1) the mission need statement and (2) the cost estimates. In addition, one of these reviews is not an independent review because it is conducted by the program office, which is on the project’s chain of command. Table 5 lists these required reviews. DOE’s requirements do not meet the other best practices in this category because DOE’s Order 413.3B does not make any reference to defining selection criteria based on the mission need; weighting selection criteria to reflect the relative importance of each; or comparing the alternatives considered, using net present value. Table 6 and appendix IV summarize our assessment of DOE’s requirements. DOE’s Requirements Combined with Guidance Do Not Conform to Best Practices DOE’s Order 413.3B AOA requirements combined with guidance partially meet best practices. Therefore, DOE does not have assurance that applying its requirements combined with its guidance may lead to reliable AOAs. DOE’s requirements combined with guidance fully or substantially met best practices in the identifying alternatives and selecting a preferred alternative categories, but not in the general principles or analyzing alternatives categories. DOE’s requirements combined with guidance conform to 9 of 24 best practices. Our assessment of DOE’s requirements combined with guidance found the following: General principles. DOE’s requirements combined with guidance partially meet best practices for the general principles category by fully or substantially meeting 3 of the 8 best practices in this category, partially or minimally meeting 3, and not meeting the other 2. DOE’s guidance supplements the requirements discussed in the previous section by suggesting that the Federal Project Director and the Integrated Project Team conduct different parts of the AOA, but the guidance does not suggest specific areas of expertise to be represented on the team. The guidance also suggests that a study plan be developed but does not suggest what to include in that plan. Neither DOE’s requirements nor guidance refer to appropriate AOA time frames or conducting the analysis without a predetermined solution. Identifying alternatives. DOE’s requirements combined with guidance substantially meet best practices for the identifying alternatives category by fully meeting 1 of the 4 best practices in this category and partially or minimally meeting the other 3. DOE’s guidance supplements the requirements by suggesting that at least three alternatives be considered, including one representing the status quo, and by listing the advantages and disadvantages of each alternative. Additionally, DOE’s guidance suggests pre-screening the alternatives for viability, but the guidance does not suggest documenting the reasons for eliminating any alternatives. Analyzing alternatives. DOE’s requirements combined with guidance partially meet best practices for the analyzing alternatives category by fully or substantially meeting 3 of the 8 best practices in this category, partially or minimally meeting 3, and not meeting the other 2. DOE’s guidance supplements the requirements by suggesting that life-cycle cost estimates be developed for each of the alternatives, the life-cycle cost estimates be presented as ranges instead of point estimates, life-cycle benefit estimate be developed for each alternative, risk be taken into consideration when evaluating alternatives, and life-cycle cost estimates be adjusted for risk. Neither DOE’s requirements nor guidance refer to presenting life-cycle cost estimates in present value terms, explaining how each measure of benefit supports the mission need, or testing the sensitivity of the cost and benefit estimates to changes in key assumptions. Selecting a preferred alternative. DOE’s requirements combined with guidance substantially meet best practices for the selecting a preferred alternative category by fully or substantially meeting 2 of the 4 best practices in this category and partially or minimally meeting the other 2. DOE’s guidance supplements the requirements by suggesting that a variety of discriminators are considered when selecting a preferred alternative, the selection criteria are weighted to reflect the relative importance of each, and the alternatives are compared using net present value. The guidance also suggests another review of more aspects of the AOA process, such as whether the alternative selection process evaluates a range of appropriate attributes for each alternative including cost, maintainability, safety, technology requirements, risks, and regulatory requirements, but the guidance suggests that this review be performed by the program office and not by a party independent of the project. Table 7 below and appendix IV summarize our assessment of DOE’s requirements combined with guidance. DOE and NNSA officials told us that unreliable AOAs are a risk factor for major cost increases and schedule delays for NNSA projects. DOE APM and NNSA APM officials acknowledged that DOE does not have definitive guidance on conducting AOAs. According to these officials, the absence of adequate guidance has led to some projects pursuing alternatives that subsequently proved to be unaffordable. For example, each of NNSA’s ongoing major projects (e.g., the Chemistry and Metallurgy Research Replacement Nuclear Facility, the Mixed Oxide Fuel Fabrication Facility, or the Uranium Processing Facility) has undergone reassessments of alternatives due to cost increases and schedule delays. As we previously reported, federal standards for internal control related to risk assessment call for agency management to assess the risks faced entity-wide and at the activity level, and that once risks have been identified, management should decide what actions should be taken to mitigate them.developing a reliable AOA process, DOE may not be successful in mitigating the risk it has identified related to this process. NNSA’s AOAs for Three Recent Projects Did Not Conform to Best Practices and Did Not Consistently Follow Certain DOE Guidance For three recent projects, our analysis found that NNSA did not conform to most of the best practices in conducting the AOAs,concerns about the reliability of these AOAs. NNSA also did not consistently follow certain DOE optional guidance for these AOAs. NNSA’s AOAs for the Three Recent Projects Did Not Conform to Best Practices NNSA did not conform to best practices overall in conducting the AOAs for the three recent projects we examined—the High Explosive Science, Technology and Engineering Project, the Radioactive Liquid Waste Treatment Facility, and the Uranium Processing Facility, therefore, raising concerns about the reliability of these AOAs. NNSA also conducted each AOA differently. (See apps. V through VII for more detailed summaries of our assessment of the AOAs for each project.) High Explosive Science, Technology and Engineering Project AOA For the High Explosive, Science, Technology and Engineering Project, NNSA partially met best practices overall in conducting the AOA. Therefore, this AOA may not be reliable. NNSA fully or substantially met best practices in the general principles and identifying alternatives categories but not in the analyzing alternatives or selecting a preferred alternative categories. NNSA conformed to 11 of 24 best practices when conducting this AOA. Our assessment of NNSA’s AOA for this project identified the following: General principles. NNSA substantially met best practices for the general principles category by fully or substantially meeting 4 of the 8 best practices in this category and by partially or minimally meeting the other 4. NNSA convened a team, which included federal and contractor project officials to conduct the analysis. Before conducting the analysis, NNSA developed a 17-step plan for conducting the AOA. When conducting the analysis, the team described in project documentation many, but not all, of the steps it took, and listed most of the assumptions made in the analysis but did not justify them. NNSA also undertook the analysis having an “originally envisioned” alternative. NNSA ultimately did not select this alternative as the preferred alternative, but NNSA did not explain how the analysis was conducted without having a predetermined solution. Identifying alternatives. NNSA fully met best practices for the identifying alternatives category by fully or substantially meeting all 4 best practices in the category. NNSA identified nine alternatives that represented a diverse range of potential solutions to the mission need, including one that represented the status quo. NNSA defined these alternatives in detail, including providing descriptions of the specific characteristics used to create cost estimates. Before proceeding to the analysis, NNSA eliminated four alternatives it did not consider viable and described the general reasons for eliminating them but did not provide specific reasons for the scores it gave each alternative as part of the screening process. Analyzing alternatives. NNSA minimally met best practices for the analyzing alternatives category by fully meeting 1 of the 8 best practices in this category, partially or minimally meeting 4, and not meeting the remaining 3. To assess the alternatives, NNSA developed cost estimates for each alternative, and presented these cost estimates with a stated accuracy range of 15 percent below to 50 percent above, but they were not life-cycle cost estimates because NNSA did not include such costs as decontamination and decommissioning in the estimates because it assumed those costs would be identical among the alternatives. NNSA did not develop quantitative estimates of the benefits that would result from each alternative, and mentioned the benefits of the alternatives only qualitatively through a discussion of some of the pros and cons of each alternative. To assess the risks associated with the alternatives, NNSA developed a list of risks for each alternative, but it did not develop potential mitigation strategies for all of the risks and did not test the sensitivity of the cost estimates to these risks or to changes in key assumptions. Selecting a preferred alternative. NNSA partially met best practices for the selecting a preferred alternative category by fully or substantially meeting 2 of the 4 best practices in this category, partially meeting 1 and not meeting 1. To select a preferred alternative, NNSA developed 21 selection criteria such as life-cycle costs, schedules, risks, flexibility, and ability to meet the mission need. NNSA then weighted these criteria on a five-point scale to reflect their relative importance, and selected the alternative that received the highest overall score. As of June 2014, NNSA planned to conduct an independent project review to assess, among other things, whether the selected preferred alternative remained the most viable and feasible alternative for meeting the approved mission need, but it did not plan on assessing other parts of the AOA process, including the identification of alternatives, the criteria used for selecting the alternative, or the benefit estimates for each alternative. Table 8 below and appendix V summarize our assessment of the AOA conducted for this project. For the Radioactive Liquid Waste Treatment Facility project, NNSA partially met best practices overall in conducting the most recent AOA in 2013. Therefore, this AOA also may not be reliable. NNSA fully or substantially met best practices in the general principles and identifying alternatives categories but not in the analyzing alternatives or selecting a preferred alternative categories. NNSA conformed to 8 of 24 best practices when conducting this AOA. Our assessment of the AOA conducted for this project identified the following: General principles. NNSA substantially met best practices for the general principles category by fully or substantially meeting 3 of the 8 best practices in this category and by partially or minimally meeting the other 5. NNSA convened a team consisting of project management and technical experts. NNSA developed detailed study documentation for some areas of the AOA process, such as for risk and cost analysis, but not for others, such as for describing how NNSA screened alternatives for viability or how NNSA developed the selection criteria. NNSA also listed but did not justify the assumptions used in the AOA report, and did not discuss any potential constraints associated with the analysis. This analysis also may have been conducted with having a predetermined solution because one of the two alternatives considered was developed from a previous design that had already incurred significant cost increases and so was unlikely to be selected. Identifying alternatives. NNSA substantially met best practices for the identifying alternatives category by fully or substantially meeting 2 of the 4 best practices in the category, and by partially or minimally meeting the other 2. NNSA identified thirty-seven options for various pieces of this project, and then combined those options into two complete alternatives to be analyzed: one that was based on the design selected in the 2006 AOA and another with a design similar to a previously-considered alternative. This approach compared an alternative developed from a design that had already incurred cost increases significant enough to warrant a reassessment of alternatives to only one other alternative. NNSA did, however, define the two alternatives in detail, assess the viability of the status quo and screen the list of thirty-seven options for viability before combining them into the two final alternatives. Analyzing alternatives. NNSA partially met best practices for the analyzing alternatives category by fully or substantially meeting 3 of the 8 best practices in this category, partially or minimally meeting 2, and not meeting 3. To assess the alternatives, NNSA developed full life-cycle cost estimates for both of the alternatives, which it presented in present-value terms, but which it did not present with a confidence interval or range. NNSA did not develop quantitative estimates of the benefits that would result from each alternative, and did not explain how any measure of benefit used would support the mission need. To assess the risks associated with the alternatives, NNSA developed a list of risks and mitigation strategies for each alternative, and NNSA adjusted the total project cost estimates for each alternative, but not the life-cycle cost estimates, for these risks. NNSA did not, however, test the sensitivity of the full life-cycle cost estimates to risks or changes in key assumptions. Selecting a preferred alternative. NNSA minimally met best practices for the selecting a preferred alternative category by partially or minimally meeting 2 of the 4 best practices in this category and by not meeting the other 2. To select a preferred alternative, NNSA compared the life-cycle cost estimates, the total project cost estimates, estimated schedules, and the scope of the alternatives. NNSA did not weight these selection criteria and did not compare the alternatives using net present value. NNSA conducted an independent project review of only the selected alternative, not of each alternative considered. NNSA also conducted a cost estimate review of total project cost estimates, not the life-cycle cost estimates, of each alternative. Table 9 below and appendix VI summarize our assessment of the AOA conducted for this project. For the Uranium Processing Facility project, NNSA partially met best practices overall in conducting the most recent AOA in 2012. Therefore, this AOA also may not be reliable. NNSA fully or substantially met best practices in the identifying alternatives category but not in the general principles, analyzing alternatives, or selecting a preferred alternative categories. NNSA conformed to 6 of 24 best practices when conducting this AOA. Our assessment of NNSA’s 2012 AOA for this project identified the following observations: General principles. NNSA partially met best practices for the general principles category by fully or substantially meeting 2 of the 8 best practices in this category and by partially or minimally meeting the other 6. NNSA convened a team composed of federal project management staff and contractors to conduct the AOA, and they conducted the bulk of the analysis over 3 days in January 2012. NNSA did not create a detailed plan for conducting the AOA. Additionally, NNSA conducted the analysis for this project with, according to NNSA officials, a fundamental assumption that the selected alternative would be a single-facility solution such as the alternative selected in the project’s previous AOA conducted in 2007. Identifying alternatives. NNSA substantially met best practices for the identifying alternatives category by fully meeting 1 of the 4 best practices in the category, and by partially or minimally meeting the other 3. NNSA identified six alternatives, all of which were variations of the single-facility solution because, according to project officials, they were “wedded” to that idea. NNSA also did not consider the status quo as an alternative. NNSA did determine that one alternative was not viable before proceeding with the analysis but did not document the screening of all the alternatives against viability criteria. NNSA described the alternatives in sufficient detail. Analyzing alternatives. NNSA minimally met best practices for the analyzing alternatives category by fully meeting 1 of the 8 best practices in the category, partially or minimally meeting 5, and by not meeting the other 2. To assess the alternatives, according to UPF project officials, NNSA developed relative cost estimates for each alternative that were updates of a 2010 total project cost estimate and that were not life-cycle cost estimates because they did not include costs that were the same across all alternatives. NNSA also did not develop quantitative estimates of the benefits that would result from each alternative. To assess the risks associated with the alternatives, NNSA listed categories of risk, such as cost, schedule, and technical risks in the description of each alternative but did not discuss specific risks or mitigation strategies. NNSA adjusted the design and construction elements of the cost estimates based on the risks identified in developing the 2010 total project cost estimate, but NNSA did not document these risks in project documentation and did not test the sensitivity of the estimates to changes in key assumptions. Selecting a preferred alternative. NNSA partially met best practices for the selecting a preferred alternative category by fully or substantially meeting 2 of the 4 best practices in this category and by partially or minimally meeting the other 2. To select a preferred alternative, according to UPF project officials, NNSA used commercially available software to compare each alternative to every other alternative. NNSA used six selection criteria, including life-cycle cost, execution flexibility, and ability to achieve benefits from modernizing and consolidating enriched uranium processing, but NNSA did not compare the alternatives using net present value. NNSA weighted these criteria on a percentage scale and selected the alternative that scored the highest. NNSA also conducted an independent review after conducting the AOA to determine if the project was technically ready, but this was not a review of the AOA process. Table 10 below and appendix VII summarize our assessment of the AOA conducted for this project. NNSA conducted each AOA differently and applied best practices differently among the projects. For example, as mentioned above in the description of the AOAs conducted at these projects, NNSA conducted the risk analysis differently for each project. The different ways that NNSA conducted the AOAs for the projects we examined may be due to the nonprescriptive nature of the Order 413.3B requirements. NNSA project officials stated that the AOA requirements in Order 413.3B are very high level and do not prescribe specific methods to follow. According to these officials, Order 413.3B provides a very general set of requirements for what the AOA process should accomplish. For example, DOE’s Order 413.3B states that life-cycle cost assumptions should be included in the conceptual design report. The word “assumptions” leaves room for interpretation because it could be interpreted as requiring the inclusion of life-cycle cost estimates for all alternatives or as requiring a life-cycle cost estimate for only the selected alternative after completion of the AOA process. In the absence of specific requirements, each AOA team developed its own approach to conducting an AOA. For example, NNSA officials who worked on the AOA for the High Explosive, Science, Technology and Engineering Project stated that they had to develop much of the AOA process themselves and that they based their process on historical AOAs conducted at the Pantex site in Texas. Officials working on the AOA for the Radioactive Liquid Waste Treatment Facility project at Los Alamos Nuclear Laboratory told us that they also used site- specific guidance including the laboratory’s engineering standards manual as well as their own professional experience. In contrast, officials working on the Uranium Processing Facility project stated that they conducted the AOA based on guidance contained within a commercially available, off-the-shelf software suite. In addition, there may be some confusion about roles and responsibilities for leading the AOA process at NNSA. NNSA officials in the Office of Acquisition and Project Management, who are responsible for managing the construction process, stated that their office does not have lead responsibility for the AOA process. However, the NNSA Concept of Operations for the Office of Acquisition and Project Management states that, while the program offices lead the development of the mission need and functional requirements and the selection of the preferred alternatives, the NNSA Office of Acquisition and Project Management has the lead for the development of the analyses of alternatives. NNSA officials stated that they will reexamine the Concept of Operations to clarify who has responsibility for the AOA process. The officials explained that NNSA APM has begun looking at, among other things, DOE’s current directives and guidance and NNSA’s policies and procedures relating to AOA, the Office of Management and Budget’s guidance relating to AOAs, and the legislation Congress has enacted in response to other federal agencies’ AOA challenges. NNSA Did Not Consistently Follow Certain DOE AOA Guidance for Its Projects For the three AOAs we examined, NNSA consistently followed the DOE AOA requirement that conformed to best practices but did not consistently follow the elements of optional guidance that conform to best practices. As noted above, DOE has one requirement that conforms to best practices, the best practice of defining functional requirements based on the mission need. NNSA followed this requirement for all three AOAs we reviewed. Similarly, DOE has eight other elements of guidance that conform to best practices (see table 11). For the three projects we reviewed, NNSA followed from one to three of these eight elements of guidance. The specific elements of guidance NNSA followed for the AOAs differed substantially by project. Specifically, NNSA followed three elements of DOE guidance that conform to best practices for the High Explosive, Science, Technology and Engineering Facility Project AOA and followed two such elements of guidance for the Radioactive Liquid Waste Treatment Facility project and only one such element of guidance for the Uranium Processing Facility project AOAs. For example, the AOA for NNSA’s High Explosive, Science, Technology and Engineering Project followed the DOE guidance that suggests identifying and considering at least three viable alternatives, including the status quo; the AOA identified nine alternatives, including one that represented the status quo. In contrast, the 2012 AOA for the Uranium Processing Facility identified six alternatives, but none represented the status quo, and the Radioactive Liquid Waste Treatment Facility project AOA identified two alternatives. Table 11 below shows the extent to which NNSA’s AOAs for the projects we reviewed followed DOE’s guidance that conform to best practices. Conclusions The AOA process is a key first step in the project management process at which DOE and NNSA have the opportunity to put projects on the right path forward. DOE has some requirements and guidance for this process in its project management Order 413.3B and associated guides. However, DOE’s AOA requirements in Order 413.3B conform to only 1 of the 24 best practices we identified from different government and private- sector entities, and the additional suggestions provided in DOE’s guides are still not sufficient to create a reliable AOA process. The AOAs for the three recent NNSA projects we reviewed—the High Explosive Science, Technology and Engineering Project, the Radioactive Liquid Waste Treatment Facility, and the Uranium Processing Facility—did not conform to best practices overall and may therefore not be reliable. DOE and NNSA officials acknowledge that unreliable AOAs are a risk factor for major cost increases and schedule delays for NNSA projects. Federal standards for internal control related to risk assessment call for management to decide on actions to mitigate identified risks. Developing requirements that incorporate best practices would help DOE mitigate the risk for major cost increases and schedule delays by creating a framework to ensure a reliable AOA process. Developing reliable AOAs is particularly important at this time because NNSA has recently reassessed or is in the process of reassessing alternatives for all its major construction projects, after spending billions of dollars on designing and partially constructing these projects. Without a process to develop reliable AOAs, NNSA may continue on this path and continue to have limited assurance that it is selecting alternatives that best meet its mission needs and will not result in major cost increases and schedule delays in the future. Recommendation for Executive Action To minimize the risk of developing unreliable AOAs and incurring major cost increases and schedule delays on projects, we recommend that the Secretary of Energy direct DOE’s Office of Acquisition and Project Management to update its project management order requirements to incorporate best practices for conducting an AOA. Agency Comments and Our Evaluation We provided DOE with a draft of this report for its review and comment. In its written comments, reproduced in appendix VIII, DOE agreed with the report’s recommendation. In its written comments, DOE stated that DOE’s order for project management (DOE O 413.3B) will be assessed for revision following the issuance of the revision to DOE-STD1189, Integration of Safety into the Design Process, currently scheduled for November 2016. DOE stated that it will consider the AOA best practices when the policy is updated. In the interim, DOE stated that it has strengthened guidance in the area of AOA, including issuing a Lifecycle Cost Handbook, and that in fiscal year 2015 it will develop and issue an AOA Handbook that will reflect the best practices for AOAs. DOE also provided technical comments that were incorporated, as appropriate. We are pleased that DOE agreed with our recommendation and that it will take action to issue an AOA Handbook. However, while an AOA Handbook may be a useful interim measure, the unspecified, open-ended date for updating the project management order that contains requirements (i.e., sometime after November 2016) and the statement that the AOA best practices will be considered, not incorporated, may indicate DOE’s lack of urgency in implementing this recommendation. We are sending copies of this report to the appropriate congressional committees, the Secretary of Energy, and other interested parties. In addition, the report is available at no charge on the GAO website at http://www.gao.gov. If you or your staff have any questions about this report, please contact me at (202) 512-3841 or trimbled@gao.gov. Contact points for our Offices of Congressional Relations and Public Affairs may be found on the last page of this report. GAO staff who made major contributions to this report are listed in appendix IX. Appendix I: Scope and Methodology To identify and describe best practices for the analysis of alternatives (AOA) process, we first searched for a source of generally accepted best practices that we could use as criteria for comparison with the Department of Energy’s (DOE) AOA process, but we could not identify a single source that was broadly recognized by government and private- sector entities and that could serve as the definitive source of best practices for the AOA process. In the absence of such a definitive source, we identified AOA handbooks, guidebooks, requirements, and other AOA- related information from federal and private-sector entities. We also sent a request to subject-matter experts, including experts from DOE, the National Nuclear Security Administration (NNSA), and various other government or private-sector entities, to help us identify further relevant information. These subject-matter experts are part of GAO’s Projects Controls Expert Working Group, which contains members with expertise in program and project management, capital acquisition, cost estimation, risk and sensitivity analysis, earned value management and scheduling, and represent a diverse range of government and private-sector entities. We reviewed the information related to the AOA process, including information from our Cost Estimating Guide,Advancement of Cost Engineering International, Department of Defense, Department of Homeland Security, National Aeronautics and Space Administration, Office of Management and Budget, United States Air Force, and a private-sector entity that specializes in acquisition management. We compiled a draft set of best practices commonly mentioned across these different entities’ AOA policies and guidance, and we sent this draft set of AOA best practices to the experts for review in advance of our semiannual meeting of the Projects Controls Expert Working Group that took place in March 2014. More than 90 experts participated, including officials from DOE and NNSA. We received comments from some of these experts both during this meeting and by e- mail after the meeting. We developed a final set of 24 best practices for the AOA process based on the comments received. We grouped these best practices into four categories: (1) general principles, (2) identifying alternatives, (3) analyzing alternatives, and (4) selecting a preferred alternative. the Association for the To determine the extent to which DOE’s requirements and guidance for conducting an AOA conform to AOA best practices, we first identified the process NNSA is required to follow when conducting an AOA. To do so, we reviewed DOE’s project management order and its associated guides, as well as other documentation received from NNSA’s Office of Acquisition and Project Management, including a compilation of DOE’s requirements and guidance related to conducting an AOA. We also interviewed officials from DOE’s Office of Acquisition and Project Management, NNSA’s Office of Acquisition and Project Management, NNSA’s Office of Cost Estimating and Program Evaluation, DOE’s Office of Science, and a former DOE official familiar with DOE’s and NNSA’s project management process, as well as NNSA and contractor officials from the programs and projects we selected for review. We then confirmed DOE’s requirements and guidance with DOE and NNSA officials, from whom we received technical comments. We performed two sets of analyses: we compared the best practices to (1) DOE’s requirements and (2) DOE’s requirements combined with the guidance. We used a five-point scoring system to determine the extent to We first used this which DOE’s AOA process conforms to best practices.scoring system to determine how well DOE’s requirements and guidance conform to each best practice. We then used the average of the scores for the best practices in each of the four categories to determine an overall score for each category, and we then used the average of the scores for the four categories as the final score for the overall DOE AOA process. If the score for each best practice, the average score for each category, or the final score for the AOA process was “fully met” or “substantially met,” we concluded that the AOA process conformed to best practices and therefore could be considered reliable. In contrast, if the score was “partially met,” “minimally met,” or “not met,” we concluded that the AOA process did not conform to best practices and therefore could not be considered reliable. For us to consider the AOA process reliable, the entire AOA process had to receive an average score of “fully met” or “substantially met,” and each individual category—(1) general principles, (2) identifying alternatives, (3) analyzing alternatives, and (4) selecting a preferred alternative—had to receive an average score of “fully met” or “substantially met” to ensure that the agency adequately and consistently performed all parts of the AOA process. GAO does not weight its cost estimating, scheduling, earned value management and AOA best practices because it is not possible to quantitatively determine the relative weights of each of the criteria. Weighting has the potential to vary across programs, as well as due to where a program may be in its different stages of its life cycle. Therefore the fairest standardized methodology to evaluate programs against is to have all criteria weighted equally. To determine the extent to which NNSA conformed to best practices in conducting the AOAs for recent NNSA projects, we used as criteria the best practices identified as part of this engagement. We chose projects for which NNSA had completed the AOA process since November 2010, when DOE’s most recent version of the project management order came into effect, or for which NNSA was scheduled to complete the AOA process by the end of fiscal year 2014. We chose projects for which NNSA had completed, or was nearing completion of, an AOA process under this version of the order because this version included significant revisions, including more specific and detailed requirements for conducting an AOA. We also selected NNSA projects that were scheduled to complete an AOA by the end of fiscal year 2014 because most of the AOA documentation would be available for these projects. We identified these projects by examining DOE’s data from its February 2014 Monthly Project Portfolio Status Report. The three projects listed in this report that met our criteria were: (1) the High Explosive Science, Technology and Engineering Project at NNSA’s Pantex site in Texas, for which NNSA was scheduled to finish the AOA process by the end of September 2014 at the time of our project selection; (2) the Radioactive Liquid Waste Treatment Facility at NNSA’s Los Alamos National Laboratory in New Mexico, for which NNSA completed its most recent reassessment of alternatives in September 2013; and (3) the Uranium Processing Facility at NNSA’s Y-12 site in Tennessee, for which NNSA completed its most recent reassessment of alternatives in June 2012. We checked with knowledgeable DOE and NNSA officials, reviewed recent GAO reports, and compared the data in the project status report with information received from NNSA on the projects to assess the reliability of the data in this status report, and we determined that the data were sufficiently reliable to ensure these projects met our criteria. For each of these projects, we reviewed project documentation, and we interviewed NNSA and contractor officials in charge of the AOA to compare the process followed by NNSA with best practices. To score each AOA process, (1) two GAO analysts separately examined the AOA documentation received from the agency and then agreed on a score for each of the 24 best practices, then, (2) an AOA specialist independent of the engagement team reviewed the AOA documentation and the scores assigned by the analysts for accuracy and cross-checked the scores in all the analyses for consistency. After completing this process for each individual best practice in all the analyses, we calculated the scores for each category—(1) general principles, (2) identifying alternatives, (3) analyzing alternatives, and (4) selecting a preferred alternative—and the overall score for each analysis. We sent our analyses to DOE and NNSA for review twice, and we used the same scoring process when we revised the scores based on their technical comments and any additional evidence received. In April 2014, as we were conducting our engagement, NNSA released a peer review report that recommended a new alternative for the Uranium Processing Facility. We examined whether this peer review reflected any characteristics of an AOA by interviewing NNSA officials and comparing the peer review with the AOA best practices. In addition, as part of this objective, we determined the extent to which NNSA conformed to certain DOE requirements and guidance—those DOE requirements and guidance that conformed to best practices—in conducting the AOAs for these projects. We used the results of the earlier analysis comparing DOE’s requirements and guidance to best practices to determine which DOE requirements and guidance conformed to best practices. We then reviewed project documentation and interviewed NNSA and contractor officials in charge of the AOA process to assess the extent to which NNSA projects followed DOE’s requirements and guidance that conformed to best practices. We conducted this performance audit from February 2014 to December 2014 in accordance with generally accepted government auditing standards. Those standards require that we plan and perform the audit to obtain sufficient, appropriate evidence to provide a reasonable basis for our findings and conclusions based on our audit objectives. We believe that the evidence obtained provides a reasonable basis for our findings and conclusions based on our audit objectives. Appendix II: The Department of Energy’s Analysis of Alternatives Process The Department of Energy’s (DOE) analysis of alternatives (AOA) process includes the following requirements from DOE Order 413.3B and suggested guidance from seven guides associated with this order. The source of the requirements or guidance is listed in parentheses at the end of the item. The Integrated Project Team—the team supporting the Federal Project Director to deliver a capital asset project and conduct the AOA—consists of professionals representing a diverse range of disciplines and specific knowledge to successfully execute the project. (O 413.3B p. C-11) As part of the preconceptual design (CD-0) approval process: The conceptual design must develop the scope required to satisfy the program’s mission requirements and identify requirements and features. A Mission Need Statement document is developed to identify the mission need—the gap that needs to be addressed—and functional requirements—the general parameters that the selected alternative must have to address the mission need. (O 413.3B pp. A-4, C-5) The mission need must be independent of a particular solution. (O 413.3B p. A-4) The program office responsible for the capital asset project must identify a credible performance gap between its current capabilities and capacities and those required to achieve the goals articulated in its strategic plan. (O 413.3B p. A-4) The program office is afforded the flexibility to explore a variety of solutions and not limit potential solutions. (O 413.3B p. A-4) As part of the conceptual design (CD-1) approval process: The conceptual design must develop reliable cost and schedule range estimates for the alternatives considered. The order also requires that whatever figure or range that is provided at the CD-0 and CD-1 stages must explicitly note relevant caveats concerning risks and uncertainties inherent in early estimated given the immature requirements definition at these stages. (O 413.3B pp. C-5, C-16) The conceptual design also must develop an assessment of project risks and identification of appropriate risk handling strategies. (O 413.3B p. C-4) Develop a conceptual design report that includes, among other things, a clear and concise description of the alternatives analyzed, the basis for the selected alternative, how the selected alternative meets the mission need, the functions and requirements that define the alternative and demonstrate the capability that the alternative can be successful, and life-cycle cost assumptions. (O 413.3B pp. A-7, C-5) The order also requires that: The project’s sponsor must never be the sole cost estimator at any stage, and that the second cost estimator must be from outside the chain of command, to avoid conflict of interest. (O 413.3B pp. A-1, C-16) Conduct several independent reviews during CD-0 and CD-1 approval processes, depending on the estimated cost of the project, related to two aspects of the AOA process: the validation of the mission need statement and of the cost estimates. (O 413.3B pp. A-7, C-7, C-17, C-18) The selected alternative must be the optimum solution that must provide the essential functions and capabilities at an optimum life-cycle cost, consistent with required cost, scope, schedule, performance, and risk considerations. (O 413.3B pp. A-2, A-5) Additionally, DOE’s order requires that as a project moves toward approval of CD-2, if the top end of the approved CD-1 cost range for the selected alternative grows by more than 50 percent, the program must reassess alternatives. (O 413.3B p. A-6) The order also requires as part of CD-1 approval that an analysis of alternatives must be conducted under the National Environmental Policy Act of 1969 (NEPA). (O 413.3B p. A-7) Summarize a planned approach to conduct an analysis of alternatives. (G 413.3-17 p. 4) The guides provide numerous examples of optional tasks for the Integrated Project Team and the Federal Project Director to perform as part of the AOA process, such as identifying functional requirements, evaluating alternatives for satisfying the requirements, conducting the appropriate analyses, and recommending a preferred alternative. (G 413.3-1 pp. 5, 7, 14, 15) (G 413.3-5A p. 8) (G 413.3-13 pp. 2, 3, 17) (G 413.3-18A p. 8) (G-413.3-21 p. 9) Explore concepts and consider alternatives for meeting the mission need until a set of viable, affordable, and sustainable alternatives is reached; consider at least three viable alternatives for analysis, including one that represents the status quo; and develop and list the primary advantages and disadvantages for each alternative. (G-413.3-13 p. 9, G 413.3-21 p. B-2) Develop cost estimates prior to the CD-2 approval milestone that are explicit ranges instead of point estimates, including life-cycle cost estimates of likely alternatives being considered that have a broad accuracy range from a low of -20 to -50 percent to a high of +30 to +100 percent. (G 413.3-21 p. 15) Quantify the benefits of alternatives over their life cycle. (G 413.3- 13 p. 10) Adjust life-cycle cost and benefit estimates for risk to ensure consideration of the alternative with the best cost-benefit ratio and generally the lowest life-cycle cost to the federal government. (G 413.3-21 p. 9) Consider various selection criteria for the alternatives, including cost and schedule, funding and budget, and technology and engineering. (G 413.3-13 p. 9) Weight the selection criteria used to select a preferred alternative for relative importance and compare alternatives using net present value or annuities. (G 413.3-13 p. 10; G 413.3-21 p. 69) At the end of the analysis, the AOA team presents the recommended alternative based on the preceding analysis in an integrated form, summarizing why an alternative is preferred, and supporting the recommendation of the preferred alternative with facts from the analysis. (G 413.3-13 p. 13) The program office performs an alternative selection and cost- range review to assess whether the AOA process (1) evaluates a range of appropriate attributes for each alternative, including cost, risks, safety, technology, and regulatory requirements and (2) is reasonable and provides best value to the federal government. (G 413.3-9 pp. 16-19) Appendix III: Best Practices for the Analysis of Alternatives Process We identified 24 best practices for identifying, analyzing, and selecting alternatives. These practices can be applied to a wide range of activities in which an alternative must be selected from a set of possible options, and to a broad range of capability areas, projects, and programs. These practices provide a framework to ensure that entities consistently and reliably select the alternatives that best meet mission needs. We grouped these 24 best practices into four categories: (1) general principles, (2) identifying alternatives, (3) analyzing alternatives, and (4) selecting a preferred alternative. The four categories of best practices address the entire analysis of alternatives (AOA) process from defining the mission need and functional requirements to independently reviewing its results. On the basis of our reviews and experts’ comments, we believe that these best practices can be generally applied from the beginning of the AOA process with practices from the general principles category, through practices in the identifying and analyzing alternatives categories, and ending with practices in the selecting a preferred alternative category. We also believe that these best practices do not necessarily have to be followed in order and that some of them can be applied concurrently with other best practices. For example, the best practice of defining the selection criteria based on the mission need in the selecting a preferred alternative category could be addressed at the same time as the best practice of creating a study plan in the general principles category. Table 12 below provides a complete list of best practices. Appendix IV: DOE’s AOA Requirements and Guidance Compared with Best Practices Overall, the Department of Energy’s (DOE) requirements for analysis of alternatives (AOA) in Order 413.3B minimally meet the best practices we identified. DOE’s requirements combined with its guidance in the guides associated with the order partially meet the best practices. Table 13 below describes our analysis of DOE’s AOA requirements and guidance compared with best practices. Appendix V: NNSA’s Analysis of Alternatives Conducted at the High Explosive, Science, Technology and Engineering Project Compared with Best Practices Overall, the National Nuclear Security Administration’s (NNSA) analysis of alternatives (AOA) conducted for the High Explosive Science, Technology and Engineering Project partially met best practices. The mission need for this project—to replace aging high explosive facilities at NNSA’s Pantex site in Texas—was approved in November 2011. NNSA expects to complete the AOA process by obtaining CD-1 in December 2014. As of September 2014, NNSA estimated that the total project cost would range from $100 to $155 million at an 85 percent confidence level. NNSA expects to finalize the AOA process by the end of 2014. Table 14 below compares the AOA conducted at the High Explosive, Science, Technology and Engineering Project with AOA best practices. Appendix VI: NNSA’s 2013 Analysis of Alternatives Conducted at the Radioactive Liquid Waste Treatment Facility Compared with Best Practices Overall, the National Nuclear Security Administration’s (NNSA) analysis of alternatives (AOA) conducted for the Radioactive Liquid Waste Treatment Facility project partially met best practices. The mission need for this project—to replace the current, aging facility—was approved in October 2004. NNSA approved an initial AOA for this project in 2006, and after substantial cost increases, conducted a second AOA (analyzed here) in 2013. NNSA currently estimates the project will cost between $168 million and $220 million. Table 15 below compares the AOA conducted at the Radioactive Liquid Waste Treatment Facility with AOA best practices. Appendix VII: NNSA’s 2012 Analysis of Alternatives Conducted at the Uranium Processing Facility Compared with Best Practices Overall, the National Nuclear Security Administration’s (NNSA) analysis of alternatives (AOA) conducted for the Uranium Processing Facility (UPF) project partially met best practices. The mission need for this project—to consolidate and replace existing enriched uranium processing capabilities at NNSA’s Y-12 site—was approved in 2004. NNSA conducted a first AOA for this project in 2007, and after substantial cost increases, conducted a second AOA (analyzed here) in 2012. NNSA estimated the project will cost between $4.2 billion and $6.5 billion. Table 16 below compares the AOA conducted at the Uranium Processing Facility with AOA best practices. Appendix VIII: Comments from the Department of Energy Appendix IX: GAO Contact and Staff Acknowledgments GAO Contact Staff Acknowledgments In addition to the individual named above, Daniel Feehan, Assistant Director; Bethany Benitez; Antoinette Capaccio; Jennifer Echard; Alex Fedell; Cristian Ion; Kirsten Lauber; Jennifer Leotta; Cynthia Norris; Karen Richey; Dan C. Royer; and Kiki Theodoropoulos made key contributions to this report. | Summary: NNSA manages numerous programs that require the design and construction of one-of-a-kind facilities. NNSA's selection of preferred alternatives for these projects is governed by DOE requirements and guidance related to conducting an AOA. In recent years, NNSA has incurred substantial cost increases and schedule delays for such projects. GAO was mandated to review the AOA process applied by NNSA. This report (1) identifies and describes AOA best practices, (2) determines the extent to which DOE requirements and guidance for conducting an AOA conform to AOA best practices, and (3) determines the extent to which NNSA conformed to best practices and followed certain DOE requirements and guidance in conducting the AOAs for recent NNSA projects. To do this work, GAO examined relevant AOA guidance from the public and private sectors and DOE's AOA requirements and guidance, sought input from AOA experts, and interviewed agency officials. GAO reviewed three AOAs that NNSA completed or was scheduled to complete between November 2010 and September 2014. GAO identified 24 best practices for analysis of alternatives (AOA)-a process that is a key first step in capital asset acquisition. The process entails identifying, analyzing, and selecting a preferred alternative to best meet the mission need by comparing the operational effectiveness, costs, and risks of potential alternatives. Because no single set of best practices for AOAs was broadly recognized by government and private-sector entities, GAO developed a set of practices by reviewing AOA policies and guidance used by seven public and private-sector entities with experience in the AOA process, and verified these practices with subject matter experts. These best practices include, among other things, defining functional requirements based on mission need, conducting the AOA without a predetermined solution, including the status-quo alternative, and conducting an independent review of the entire AOA process. These practices can be applied to a wide range of activities, projects, and programs. The Department of Energy's (DOE) requirements and guidance-found in the agency's orders and associated guides-for conducting an AOA do not conform to the 24 best practices GAO identified. Therefore, DOE does not have assurance that applying these requirements and guidance may lead to reliable AOAs. GAO's review of DOE's requirements for AOAs found that they conform to only 1 of the 24 best practices: the practice of defining functional requirements based on mission need. GAO's review of DOE's requirements combined with associated guidance-which includes nonmandatory approaches for meeting requirements-found that they conform to 9 of the 24 best practices. For example, DOE's guidance suggests identifying and considering at least three viable alternatives, including the status quo-a best practice that is not included in the requirements. Federal standards for internal control related to risk assessment call for agency management to decide on actions to mitigate identified risks. Without developing a reliable AOA process, DOE may not be successful in mitigating the risk it has identified related to this process. DOE's requirements and guidance for AOAs also apply to the National Nuclear Security Administration (NNSA), a separately organized agency in DOE. For three recent NNSA projects that GAO reviewed, NNSA did not conform to most of the best practices GAO identified for conducting AOAs, therefore, raising concerns about the reliability of these AOAs. Specifically, for the three projects' AOAs, NNSA conformed to 6, 8, and 11 of the 24 identified best practices, respectively. For all three projects, NNSA consistently followed the one DOE requirement that conformed to a best practice-to define functional requirements based on mission need. DOE and NNSA officials acknowledge that unreliable AOAs are a risk factor for major cost increases and schedule delays for NNSA projects. As GAO has previously reported, NNSA has spent billions of dollars designing and partially constructing projects with an estimated cost of $750 million or more, only to later reassess alternatives. Without a process to develop reliable AOAs, NNSA may continue on this path and continue to have limited assurance that it is selecting alternatives that best meet its mission needs and will not result in major cost increases and schedule delays in the future. | 14,896 | 940 | gov_report | en |
Summarize: These crawls are part of an effort to archive pages as they are created and archive the pages that they refer to. That way, as the pages that are referenced are changed or taken from the web, a link to the version that was live when the page was written will be preserved.Then the Internet Archive hopes that references to these archived pages will be put in place of a link that would be otherwise be broken, or a companion link to allow people to see what was originally intended by a page's authors.The goal is to fix all broken links on the web. Crawls of supported "No More 404" sites. Despite successful ACL repair surgery and rehabilitation, some patients with ACL-repaired knees continue to experience so-called 'pivot shift', or episodes where the knee 'gives way' during activity. For the last four years, orthopaedic surgeons Dr Steven Claes and Professor Dr Johan Bellemans have been conducting research into serious ACL injuries in an effort to find out why. Their starting point: an 1879 article by a French surgeon that postulated the existence of an additional ligament located on the anterior of the human knee. That postulation turned out to be correct: the Belgian doctors are the first to provide a full anatomical description of the ligament after a broad cadaver study using macroscopic dissection techniques. Their research shows that the ligament, called the anterolateral ligament (ALL), was noted to be present in all but one of the 41 cadaveric knees studied. Subsequent research shows that pivot shift, the giving way of the knee in patients with an ACL tear, is caused by an injury in the ALL ligament. Some of the researchers' conclusions were recently published in the Journal of Anatomy. The Anatomical Society praised the research as "very refreshing" and commended the researchers for reminding the medical world that, despite the emergence of advanced technology, our knowledge of the basic anatomy of the human body is not yet exhaustive. The research questions current medical thinking about serious ACL injuries and could signal a breakthrough in the treatment of patients with serious ACL injuries. Dr Claes and Professor Bellemans are currently working on a surgical technique to correct ALL injuries. Those results will be ready in several years. ACL tears are common among athletes in pivot-heavy sports such as soccer, basketball, skiing and football. If you thought thousands of years of scholarship had illuminated all human anatomy, think again. In a new study from University Hospitals Leuven in Belgium, researchers describe the first-ever dissection of the anterolateral ligament – an entirely “new” body part located within the muscles and tendons of the knee. The findings may help physicians and orthopedic surgeons develop more sophisticated treatment methods for anterior cruciate ligament (ACL) tears and other common knee injuries. The existence of an unknown knee ligament was first postulated by the French surgeon and knee expert Paul Segond, who in 1879 authored a paper implicating a hypothetical “pearly, resistant, fibrous band” in certain fractures. More than a century later, the ligament continues to puzzle researchers, as it has consistently evaded a uniform definition. The current study, which used Segond’s paper as a point of departure, sought to determine once and for all whether this mysterious structure is in fact a part of gross human anatomy. To investigate, lead author Steven Claes and his team performed macroscopic dissections of 41 cadaveric knees. In all but one of the knees, the researchers found that the “pearly band” was a well-defined, clearly distinguishable structure connecting the femur to the anterolateral tibia. The structure, dubbed the “anterolateral ligament” (ALL), is located in the so-called anterolateral aspect of the knee. According to the researchers, the existence of the ALL helps explain why patients with ACL injuries continue to experience complications after surgery and treatment. For example, many complain about “pivot shifts” that cause their damaged knee to give way. “By providing a detailed anatomical characterization of the ALL, this study clarifies the long-standing enigma surrounding the existence of a ligamentous structure connecting the femur with the anterolateral tibia,” the study authors wrote in their conclusion. “Given its structure and anatomic location, the ALL is hypothesized to control internal tibial rotation and thus to affect the pivot shift phenomenon, although further studies are needed to investigate its biomechanical function.” The ALL is not the only new addition to next year’s anatomy books. In July, a similar research effort identified an entirely new layer of the human cornea. According to the authors, the discovery of “Dua’s layer” will make future eye surgery much safer. Like the current study, “Human Corneal Anatomy Redefined: A Novel Pre-Descemet's Layer (Dua's Layer)” reminds us that the human body still harbors profound secrets. Source: Steven Claes, Evie Vereecke, Michael Maes, Jan Victor, Peter Verdonk, Johan Bellemans. Anatomy of the anterolateral ligament of the knee. Journal of Anatomy, 2013. | Summary: Two Belgian surgeons have discovered a new body part-though there's potentially a 3% chance you don't actually have it. In a paper published in the Journal of Anatomy, they confirm the existence of the knee's anterolateral ligament. They were far from the first to speculate on the mysterious "enigmatic" ligament, though: That honor goes to Frenchman Paul Segond, who wrote an 1879 paper that speculated about the "pearly, resistant, fibrous band," reports Medical Daily. To put Segond's theory to the test, Dr. Steven Claes and Dr. Johan Bellemans dissected the knees of 41 cadavers. Science Daily reports they found the ALL-which sits toward the front and side of the knee, and connects the femur and anterolateral tibia-in 40, or 97%, of them. (You can see a somewhat grisly photo here.) But the news isn't just neat, it's potentially quite useful: The surgeons say their findings help explain why those who have treated an ACL injury still have issues, specifically experiencing "pivot shifts," in which the knee gives way. Based on the ALL's structure and location, the doctors assert that an ALL injury may be what causes the pivot shift. The men plan to next develop a method of repairing injured ALLs. | 1,138 | 306 | multi_news | en |
Summarize: FIELD OF INVENTION [0001] The present invention relates to a jewelry cover prevents jewelry from abrading people and surfaces it touches, and protects exposed jewelry articles from foreign substances and damage. More specifically, the present invention provides a flexible, durable, comfortable, disposable, and cost effective strip that is easily applied to and removed from said jewelry article and the user's appendage. BACKGROUND OF THE INVENTION [0002] Rings, jewelry, bands, and similar accessories are all constantly exposed to the elements and messy substances. Jewelry articles worn on all appendages, including toe-rings and other foot-worn decorations, are susceptible to harmful substances in addition to routine wear and tear. Substances can damage jewelry, thus negatively affecting its value, as well as damage its cosmetic appeal. [0003] Further, jewelry can inadvertently harm people and items, as well as puncture or damage gloves or other clothing. Innocuous contact with another can potentially lead to a hurtful scratch or cut from a sharp edge of a jewelry article. People employed in jobs requiring frequent use of hands and physical contact, such as physical therapists or dental hygienists, risk harming a patient every time they touch. These medical professionals may place latex gloves over their jewelry, but sharp edges can snag and pierce the gloves, rendering them useless. Further, tools and utensils are subject to scratching, abrasions, and similar damage whenever someone wearing jewelry uses them. As users continuously contact others there is an ever-growing need for a cover to protect the jewelry and similar accessories. Although there are many covers known in the art for use in protecting jewelry articles, none resolve all of the problems solved by the present invention. [0004] For instance, there are many known casings that specifically cover finger-worn articles. Although these casings are protective, they are rigid and typically made of hard plastics. These hard materials hinder flexibility, thus restricting movement of the hand or finger. Due to their rigidity and inflexibility, hard casings are uncomfortable and even abrasive. [0005] Other covers made of more pliable materials can also restrict movement. These pliable covers may not secure to an appendage as well as covers made of rigid materials. To compensate for this lack of stability, known pliable covers wrap around several appendages even though the jewelry intended to be protected fits on a single digit. For instance, there are known jewelry covers that teach leather straps wrapped around a finger to protect a ring, but further include a separate element worn around the user's wrist or palm. A support element then connects the finger wrap and wrist or palm wrap elements. Ultimately, three (and sometimes more) strap pieces combine in the same cover device to provide the necessary stability. These cumbersome covers and may not provide the same level of protection as their rigid counterparts and yet still limit the user's movement. [0006] Additional covers known in the art have similarly complicated assemblies. Many covers stay in place by chains connecting the cover to a wristband element. These chains are typically structurally weak; should any one link snap, the cover will detach and expose the jewelry. Therefore, even more intricate jewelry covers do not lead to enhanced jewelry protection. The more parts a cover has, the more it may restrict movement, and the more difficult it may be to apply to an appendage. Jewelry covers having fewer structural and interrelated elements are likelier to result in easy-on, easy-off application as opposed to those with a greater number of parts. [0007] Further, as the number of parts increases, so does the cost of the cover. Fewer components leads to cheaper manufacturing costs and allows suppliers to provide an inexpensive product to the consumer. Reducing the number of elements lessens the need for repair and maintenance opportunities, therefore lowering future costs as well. [0008] Many covers intended for multiple uses accrue dirt and remnants of harmful substances. Constant cleaning of the cover can be costly and abrasive cleaners may wear away protective coverings. Cursory cleanings allow residue of the harmful substance to remain on the cover. Unclean covers can harbor germs and lead to dirtier, more harmful substances than the very materials the covers intend to protect against. Disposing of the cover after a finite number of uses prevents unsanitary covers from contacting jewelry and spreading to the user. Many of the covers already described, especially those made of costlier materials like leather, or those with elaborate chain elements, are meant for repeated uses. Disposable covers have a far greater chance of promoting sanitary conditions than nondisposable ones. [0009] Many jewelry covers in the art have the disadvantage of being specifically tailored to articles worn on the finger or wrist. Usually, jewelry covers protect rings and precious gems lodged in finger or hand worn articles but are not adapted to protect articles worn on other appendages. Covers employing multiple strap elements, chains, or hard plastics are cumbersome enough to use on a hand, let alone a toe, ankle, or foot. Toe-rings and foot-worn jewelry must remain exposed and cannot receive the protective benefits of known jewelry covers. [0010] Another disadvantage of known jewelry covers, especially those made of hard materials, is their tendency to damage additional layers of clothing. A hard jewelry cover may pierce a latex glove intended to be worn over the jewelry cover. Covers with chains or other protruding elements may catch, puncture, and/or tear fabric gloves or socks. When a user puts on a jacket or shirt, known covers can catch on to the fabric and damage the clothing or fall off from the appendage and expose the jewelry. [0011] Therefore, there is a need in the art for a jewelry cover that protects people and articles from scratches, damage, and general harm; does not damage, pierce, puncture, snag, or tear other articles of clothing; protects jewelry articles from unwanted substances; is made of a durable, flexible material that can be worn on any appendage; provide easy-on, easy-off application and detachment; is comfortable to the wearer; is cost-effective; and is disposable. SUMMARY OF THE INVENTION [0012] In order to meet the need in art for a jewelry cover that protects people and articles from scratches, damage, and general harm; does not damage, pierce, puncture, snag, or tear other articles of clothing; protects jewelry articles from unwanted substances; is made of a durable, flexible material that can be worn on any appendage; provide easy-on, easy-off application and detachment; is comfortable to the wearer; is cost-effective; and is disposable, the present invention has been devised. [0013] The present invention is a flexible elongate strip adapted to wrap around a jewelry article worn on a user's appendage. In this context, an appendage includes any body part that receives a jewelry article; typically, but not limited to, a finger or a toe, and can further include a hand, palm, foot, wrist, or ankle. Jewelry articles are not limited to cosmetic decorations of great monetary or sentimental value, but rather may include any accessory worn on any appendage. [0014] The strip is substantially flat and typically does not resemble an exact geometric shape. However, the strip can be any known shape provided it wraps around an appendage. To maintain necessary flexibility and durability, the strip is typically made of silicone, but can be made of any similarly durable and flexible material that prevents the penetration of messy and/or harmful substances, including, but not limited to, rubber, plastic, and fabric. [0015] Regardless of its shape and material, the strip does not have corners or edges sharp enough to puncture a latex glove or catch on a piece of fabric clothing. Due to the strip's durability, the user can wrinkle and place it in his pocket or purse without risk of damaging, creasing, or tearing. The flexible material provides smooth, comfortable contact with the user's skin and does not hinder movement. [0016] Further, the smooth material allows the user to physically contact other people without harming them. For instance, users engaging in regular physical contact with others, such as the aforementioned physical therapists and dental hygienists, can use the jewelry cover to protect their patients from the sharp edges of jewelry articles when the present invention is in use, and at the same time protect their jewelry and prevent tearing, snagging, piercing, or puncturing of gloves. The material's flexibility also allows users to move their appendages without hindering motion, so people can work without having to deal with cumbersome jewelry covers. These are clear advantages over known jewelry covers, especially covers made of rigid materials. [0017] A typical embodiment comprises a strip having two ends, usually referred to as a first end and a second end, with both ends generally rounded at the corners. The portion of strip connecting the two ends has generally parallel straight edges. Positioned towards the first end in the preferred embodiment is a substantially hollow housing having a cross-sectional shape. This housing protrudes from the top side of the strip and is covered on all sides and on top by walls made of the same material as the strip. It has a cross-sectional shape adapted to receive at least one precious stone, gem, or similar element protruding from a jewelry article. Although bounded by walls and a top, the housing has no base; the bottom surface of the strip opens such that its inner walls are exposed. The inside surfaces of the walls and top define a cavity occupied by protruding elements from jewelry articles when the cover is worn. The housing of the preferred embodiment is adapted to receive at least one such protruding element of a jewelry article. [0018] In many embodiments, a lip surrounds the base of the housing, i.e. where the housing begins protruding from the top surface of the strip. This lip extends the cross-sectional shape of the housing past the generally parallel straight edges of the strip. To best accept all shapes and sizes of protruding jewelry elements, the housing's cross-sectional shape is substantially elliptical or oval-shaped. Further, the lip separates the first end with the straight, middle portion of the strip. [0019] Another major feature of the present invention is the securement means. The securement means secures the jewelry cover to the appendage while in use. Most embodiments employ a throughhole adapted to closely receive the housing. Even in the absence of additional securement means elements, this configuration conceals the jewelry article and still maintains a tight, comfortable fit around the appendage. [0020] The throughhole is positioned towards the second end of the strip, opposite the end in which the housing protrudes. The throughhole extends longitudinally through the entire thickness of the strip and has substantially the same cross-sectional shape as the housing. When the strip is in use, the housing covers the jewelry article; the middle portion of strip wraps around the appendage; the throughhole wraps back to the housing; and the housing extends through the throughhole. Since the throughhole is substantially the same cross-sectional shape as the housing, it too has an outer lip that extends the outer edge of the strip. This lip separates the second end of the strip with the middle portion. [0021] Many different kinds of securement means may be used to provide added stability to the strip. Snaps, buttons, adhesives, and clasps, either used individually, in combination, with the throughhole, or independent of the throughhole, all provide means of ensuring the cover has a tight, comfortable fit around the appendage. [0022] One such securement means includes at least one raised protrusion extending from the top surface of the strip, similar to how the housing extends from the top surface of the strip. This protrusion has its own cross-sectional shape. The present invention does not require that the protrusion and housing have substantially similar cross-sectional shapes. The protrusion is positioned in proximity to the housing, opposite the first end of the strip. The protrusion is preferably laterally disposed relative to the strip, i.e. oriented substantially at a right angle relative to the parallel edges of the strip. Most embodiments utilize at least one protrusion. [0023] The protrusion is adapted to fit inside an opening positioned near the second end of the strip. The opening is a hole or slit extending longitudinally through the entire thickness of the strip, similar to the throughhole. The opening is positioned between the second end of the strip and the throughhole adapted to receive the housing when the strip is in use. This contrasts from the protrusion which is not between the first end and housing, but located on the side of the housing closer to the parallel edges. [0024] There is at least one opening for every protrusion. To maximize securement, the opening has substantially the same cross-sectional shape as the protrusion, as the opening is adapted to closely receive the protrusion. When in use, the throughhole of the present invention will receive the housing, and each opening will receive exactly one protrusion. The total number of openings may exceed the number of protrusions so the user can adjust the band as needed for comfort. Although the throughhole fits over the housing, the flexible material may stretch as needed with the extra openings. The strip becomes more secure around the user's appendage and jewelry article as the number of protrusions and openings increases. [0025] The following description best describes the present invention's functionality: a user places a jewelry article on an appendage; for example, an engagement ring having at least one precious stone is placed on a finger. The housing accepts the stone such that the cavity defined by the inner surfaces of the housing envelopes the entire stone. The middle portion of strip wraps around the user's finger such that the bottom surface of the strip contacts the ring or band. The strip wraps around the entire ring leaving no portion of the ring exposed. To secure the cover in place, the throughhole is brought towards and placed over the housing. The throughhole closely receives the housing since they have the same cross-sectional shape. This configuration completely covers the engagement ring, therefore protecting it from damaging substances, as well as scratching anyone or anything. With the housing now extended through the throughhole, the second end of the strip provides added securement when at least one protrusion slots into at least one opening. [0026] In this respect, before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangements of the components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced and carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein are for the purpose of description and should not be regarded as limiting. [0027] As such, those skilled in the art will appreciate that the conception, upon which this disclosure is based, may readily be utilized as a basis for the designing of other structures, methods, and systems for carrying out the several purposes of the present invention. It is important, therefore, that the claims be regarded as including such equivalent constructions insofar as they do not depart from the spirit and scope of the present invention. These and other constructions will become obvious to those skilled in the art from the following drawings and detailed description of the preferred embodiment. BRIEF DESCRIPTION OF THE DRAWINGS [0028] FIG. 1 is a top plan view of the preferred embodiment of the present invention. [0029] FIG. 2 is a top isometric view of the preferred embodiment of the present invention. [0030] FIG. 3 is a bottom plan view of the preferred embodiment of the present invention. [0031] FIG. 4 is a bottom isometric view of the preferred embodiment of the present invention. [0032] FIGS. 5-6 are top isometric views of alternate embodiments with alternate securement means. [0033] FIG. 7 is an elevation view of the preferred embodiment of the present invention. [0034] FIG. 8 is an environmental view of the present invention in use on a user's finger. [0035] FIGS. 9-12 are top isometric views of alternate embodiments with alternate securement means. [0036] FIG. 13 is a top isometric view of an alternate embodiment without the throughhole configuration. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0037] The following detailed description and corresponding drawings are of the best currently contemplated modes of carrying out exemplary embodiments of the invention. The description is not to be taken in a limiting sense, but is made for the purpose of illustrating the general principles of the invention. [0038] Referring now to FIG. 1, a preferred embodiment of the present flexible jewelry cover invention 10 is shown. The flexible jewelry cover 10 is an elongate strip preferably made of silicone or a similarly flexible yet durable material that feels comfortable against a user's skin and will not puncture, pierce, snag, or tear additional layers of clothing placed over it. Cover 10 has a top surface 15 with an outer edge, described in detail below, which tapers outwardly and downwardly to bottom surface 60 (as shown in FIG. 3 ). [0039] At its top surface 15, the shape of the cover 10 is bounded by a first, arch-shaped edge 2 having generally rounded and tapered edges 4, and a second edge 3 having rounded corners 5. The arch-shape indicates which end the user will grip when applying the cover 10 to an appendage. The first edge 2 and its tapered edges 4 define the first portion 20 of the cover 10, and the second edge 3 having rounded corners 5 define the second portion 30. Connecting the two ends 20 and 30 is middle portion 40. Middle portion 40 is bounded on its sides by substantially parallel straight edges 6. [0040] Positioned between the first portion 20 and the middle portion 40 is housing 1. Housing 1 can have any cross-sectional shape, provided the cavity defined by the inner walls 70 and inner surface 72 of the housing cap 17 (as shown in FIG. 4 ) are adapted to accept at least one jewelry article. In the preferred embodiment, housing 1 has a substantially elliptical or oval cross-sectional shape as shown. [0041] Positioned between the second portion 30 and the middle portion 40 is throughhole 7. Throughhole 7 can have any cross-sectional shape, provided it is substantially the same cross-sectional shape as housing 1 as throughhole 7 is adapted to closely receive housing 1. In the preferred embodiment, throughhole 7 has a substantially oval cross-sectional shape as housing 1 has a substantially oval cross-sectional shape. [0042] Surrounding the housing 1 at its base is a base support 19. The base support 19 provides added durability to the housing 1 at its base, further preventing the housing 1 from tearing, shearing, or breaking off the rest of the strip. Base support 19 has generally the same cross-sectional shape as the housing 1. Surrounding the base support 19 is first outer lip 11. First outer lip 11 has generally the same cross-sectional shape as the housing 1 (and, as it follows, base support 19 ) and extends the outer boundary of the cover 10 at the first outer boundary 13. Found on either side of the strip, first outer boundary 13 bulges out past the outer dimensions of first portion 20 and middle portion 40 established by tapered edges 4 and parallel edges 6, respectively. Similarly, second outer lip 12 surrounds throughhole 7 and extends the outer boundary of the cover 10 at second outer boundary 14. Also found on either side of the strip, second outer boundary 14 bulges out past the outer dimensions of second portion 30 and middle portion 40 established by rounded corners 5 and parallel edges 6, respectively. [0043] The lips 11 and 12 and outer boundaries 13 and 14 have generally the same shape as the cross-sectional shape of housing 1 and throughhole 7, respectively. As shown in FIG. 1, first outer lip 11 is generally oval shaped and first outer boundary 13 is generally arcuate as the cross-sectional shape of housing 1 is substantially an oval. Second outer lip 12 is generally oval shaped and second outer boundary 14 is generally arcuate as the cross-sectional shape of throughhole 7 is substantially an oval. [0044] First portion 20, first outer lip 11, middle portion 40, second outer lip 12, and second portion 30 are all coplanar and past of the top surface 15. [0045] Housing 1 is bounded on all sides by a wall 16 (as shown in FIG. 2 ), said wall 16 protruding from the top surface 15. The walls 16 terminate at the housing cap 17. The cap 17 is the first element of the cover 10 to extend through throughhole 7 when the cover 10 is in use. [0046] FIG. 1 also depicts the top surfaces 46 of protrusions 45 (shown in full in FIG. 2 ). The top surfaces 46 cap off the protrusion walls 47 (shown in FIG. 2 ). In the preferred embodiment shown in FIG. 1, the cross-sectional shape of the protrusions 45 is generally elongate and oval shaped. The protrusions 45 are positioned near the housing 1 but opposite the first edge 2, and are instead oriented in the middle portion 40 between parallel straight edges 6. [0047] Shown in FIGS. 1 and 2 are the openings 50, each of which has substantially the same cross-sectional shape as the protrusions 45 as they are adapted to closely receive the protrusions 45 when the cover 10 is in use. The openings 50 are positioned on the second portion 30 between throughhole 7 and second edge 3. There is at least one opening 50 for every protrusion 45. In the preferred embodiment there is at least one protrusion 45, and as it follows, at least one opening 50. In many embodiments, both the protrusions 45 and openings 50 are laterally disposed relative to the strip, i.e. they are generally oriented at a right angle relative to the parallel edges 6 of the strip. [0048] Additional elements located on first portion 20 are grips 8. Many embodiments contain a plurality of at least one grip 8 which help the user apply the cover 10 over a jewelry article and to an appendage. Grips 8 can extend from the top surface 15, albeit it not as far as housing 1 or protrusions 45, or they may recess into the cover 10. Recessed grips 8 do not recess longitudinally through the entire thickness of the strip. Grips 8 do not need to be an exact shape; the grips 8 shown in FIG. 1 are substantially the same shape as protrusions 45. One grip 8 does not need to be the exact same shape and dimension as another. Shown in the drawings is a plurality of five grips 8. The grips 8 are arranged such that their center lines 9 that bisect them are collinear. This is not required, as any configuration or orientation that assists the user in applying the cover 10 to the jewelry article appendage is acceptable. [0049] FIG. 2 shows the preferred embodiment at an isometric angle. At this view, the walls 16 of housing 1 as well as the walls 47 of protrusion 45 are seen. Further, a clearer view of base support 19 surrounding housing 1 is seen. As shown in FIGS. 2 and 3, the shape of the cover 10 is bounded at its bottom surface by an arch-shaped, first bottom edge 62 with bottom tapered edges 64, and a second bottom edge 63 having bottom rounded corners 65. [0050] FIG. 3 depicts a plan view of the bottom surface 60 of cover 10. In addition to the elements that comprise the bottom, outer edge of the strip, FIG. 3 shows the inner surface 72 of housing cap 17. Further, FIG. 3 shows the opposite sides of openings 50 ; in this drawing, the openings 50 are not capped (see FIGS. 5 and 6 for capped openings 55 ). The opposite side of throughhole 7 is also observable. Many embodiments of the strip include the chamfered edge 74. The middle portion 61 of bottom surface 60 is bounded by substantially straight, parallel edges 66. Chamfered edge 74 tapers inwardly into the cavity and provides connection of the inner wall 70 to bottom surface 60. This connection is best seen in the isometric view of FIG. 4, in which inner surface 72, inner walls 70, and chamfered edge 74 are all viewable. [0051] FIGS. 5 and 6 depict an alternate embodiment of the present invention complete with capped openings 55. These caps have outer walls 57, similar to the outer walls 47 of the protrusions, as well as a top 56, analogous to the top 46 of the protrusions 45. These outer walls 57 extend from the second portion 30. The capped openings 55 have a cross-sectional shape substantially the same as the cross-sectional shape as the protrusions 45, and as it follows, the openings 50. The openings 50 are not sealed in the capped embodiments, but instead provide the opening through the strip that accepts the protrusions 45. These openings 50 in the bottom surface 60 are best shown in FIG. 3. FIG. 5 depicts the simplest embodiment, in which the cover 10 features one protrusion 45 and one capped opening 55. FIG. 6 shows another alternate embodiment, complete with multiple protrusions 45 and multiple capped openings 55. The more protrusions and capped openings 55, the better the securement around the jewelry article and the user's appendage. [0052] FIG. 7 provides an elevation view of the preferred embodiment of the present invention. The outer wall 16 of housing 1, protrusions 45, and the protrusion outer walls 47 are seen extending from the top surface 15. Bounding the base of housing 1 is base support 19. Along the bottom surface, first bottom edge 62 and second bottom edge 63 are seen at opposite sides of the strip. Along the top surface, first edge 2 and second edge 3 are also viewed at opposite sides of the strip. This drawing also depicts an elevation view of embodiments in which grips 8 are recessed into the strip. [0053] FIG. 8 shows the present invention while in use. Here, the cover 10 is wrapped around a user's appendage, depicted as a finger F. The cavity inside housing 1 defined by inner walls 70 and inner surface 72 completely encapsulates a jewelry article worn on finger F. The cover 10 then wraps around the support element for the jewelry article, which would be a ring or similar finger-worn accessory in this embodiment, until the support element is also covered. Throughhole 7 is wrapped around with the rest of the strip and housing 1 is inserted through it, housing cap 17 the first element of housing 1 to extend through throughhole 7. Throughhole 7 is stretched and in direct contact with outer wall 16. In this drawing, middle portion 40 is seen through a stretched portion of throughhole 7. Additional securement means are not pictured in this drawing. [0054] FIGS. 9-12 feature alternate embodiments of the present invention, each with a different securement means different than the protrusion-and-opening configuration previously described. FIG. 9 depicts a snap securement means 80 affixed to the top surface 15 of cover 10. In this drawing, snap means 80 is positioned on the side of the housing 1 where the protrusions 45 are located in the preferred embodiment. Snap means 80 can be positioned throughout anywhere middle portion 40 provided the jewelry article is protected when the cover 10 is in use. The snap insert 85 extends from the bottom surface 60 so that it can engage the snap means 80 when in use. The securing element 86 that affixes the snap insert 85 into the strip is shown protruding through second portion 30. [0055] FIG. 10 depicts a clasp 90 as the securement means, said clasp 90 positioned on the side of the housing 1 where the protrusions 45 are located in the preferred embodiment. Clasp 90 can be positioned anywhere throughout middle portion 40 provided the jewelry article is protected when the cover 10 is in use. The edge of the strip defined by second edge 3 and second bottom edge 63 is fed through the opening 95 when the cover 10 is in use. After this edge is fed through opening 95, the user closes the clasp latch 97 by backwardly rotating it about hinge 98. Once the clasp latch 97 is tight enough that it cannot move, but is still comfortable to the user, the cover 10 is secured. [0056] FIG. 11 depicts the securement means as a button 100. In this drawing, button 100 is affixed to the top surface 15 and positioned on the side of the housing 1 where the protrusions 45 are located in the preferred embodiment. Button 100 can be positioned anywhere throughout middle portion 40 provided the jewelry article is protected when the cover 10 is in use. Opening 50, previously adapted to closely receive the protrusion 45, is now adapted to wrap around button 100. When the cover 10 is in use, housing 1 will extend through throughhole 7, and the user will place opening 50 around the button 100. In this embodiment, the opening 50 is not capped. [0057] FIG. 12 depicts an adhesive 110 placed on the top surface of the strip 15 as an alternate securement means. In this drawing, adhesive 110 positioned on the side of the housing 1 where the protrusions 45 are located in the preferred embodiment. Adhesive 110 can be positioned anywhere throughout middle portion 40 provided the jewelry article is protected when the cover 10 is in use. When the strip is in use, and the housing 1 has extended through throughhole 7, the user presses the free end of the strip, i.e. the edge of the strip defined by second edge 3 and second bottom edge 63, to the adhesive 110 for securement. Pressing the strip together allows the bottom surface 60 of the strip to adhere to the top surface 15. The adhesive 110 may be resealable as needed. Alternatively, the strip itself may be self-adhesive. There may be another adhesive section 110 on the bottom surface 60 positioned where the openings 50 are located in the preferred embodiment. This extra section of adhesive would provide even greater securement when the strip is in use. The user would press the ends together in the same manner as if there were only one adhesive section. [0058] FIG. 13 provides yet another alternative embodiment which does not employ a throughhole. Instead, the cover 10 securement means utilizes an alternative securement means, such as a snap, clasp, button, or adhesive as depicted in FIGS. 9-12. For illustrative purposes, FIG. 13 shows use of the adhesive 110, wherein the adhesive 110 is positioned on the first portion 20 as opposed to being on middle section 40, i.e. the side of the housing 1 where the protrusions 45 are located in the preferred embodiment as shown in FIG. 12. [0059] Here, the housing 1 accepts at least one jewelry article and any elements extending from it, and the strip wraps around the appendage just as in the preferred embodiments. However, the far end of the strip defined by the second edge 3 and bottom edge 63 wrap around the appendage and terminate at first portion 20. The user then presses the free end of the strip to the adhesive 110 in a similar fashion as the embodiment depicted in FIG. 12. Pressing the strip together allows the bottom surface 60 of the strip to adhere to the top surface 15. Alternatively, the strip itself may be self-adhesive. With no throughhole, no part of the strip closely receives housing 1. Further, the strip may be much shorter than in other embodiments, as depicted by a condensed middle section 40. [0060] Similar to the embodiment featured in FIG. 12, the adhesive 110 may be resealable as needed. There may be another adhesive section 110 on the bottom surface 60 positioned where the openings 50 would be located in the preferred embodiment. This extra section of adhesive would provide even greater securement when the strip is in use. The user would press the ends together in the same manner as if there were only one adhesive section, or if the strip were configured in the same way as previously disclosed. [0061] It should be understood, of course, that the foregoing relates to exemplary embodiments of the invention and that modifications may be made without departing from the spirit and scope of the invention as set forth in the following claims. [0062] Therefore, the foregoing is considered as illustrative only of the principles of the invention. Further, since numerous modifications and changes will readily occur to those skilled in the art, it is not desired to limit the invention to the exact construction and operation shown and described, and accordingly, all suitable modifications and equivalents may be resorted to, falling within the scope of the invention. | Summary: A flexible, durable, comfortable, cost-effective, easy-on, easy-off flexible jewelry cover completely envelopes jewelry articles and elements extending from said articles, preventing the user from scratching, abrading, or otherwise harming other people or articles he may touch. Further, the cover protects the jewelry articles from unwanted, damaging, and messy substances. The cover may be adapted for use on any appendage, including, but not limited to, fingers, toes, wrists, ankles, hands, and feet. Due to the durable, yet flexible material, the cover fits comfortably on any appendage and does not restrict bodily movements. The cover will not pierce or puncture exterior layers of clothing such as gloves and socks. The cover is also disposable, therefore promoting sanitary conditions. | 8,145 | 180 | big_patent | en |
Summarize: WHEN another night came the columns, changed to purple streaks, filed across two pontoon bridges. A glaring fire wine-tinted the waters of the river. Its rays, shining upon the moving masses of troops, brought forth here and there sudden gleams of silver or gold. Upon the other shore a dark and mysterious range of hills was curved against the sky. The insect voices of the night sang solemnly. After this crossing the youth assured himself that at any moment they might be suddenly and fearfully assaulted from the caves of the lowering woods. He kept his eyes watchfully upon the darkness. But his regiment went unmolested to a camping place, and its soldiers slept the brave sleep of wearied men. In the morning they were routed out with early energy, and hustled along a narrow road that led deep into the forest. It was during this rapid march that the regiment lost many of the marks of a new command. The men had begun to count the miles upon their fingers, and they grew tired. "Sore feet an' damned short rations, that's all," said the loud soldier. There was perspiration and grumblings. After a time they began to shed their knapsacks. Some tossed them unconcernedly down; others hid them carefully, asserting their plans to return for them at some convenient time. Men extricated themselves from thick shirts. Presently few carried anything but their necessary clothing, blankets, haversacks, canteens, and arms and ammunition. "You can now eat and shoot," said the tall soldier to the youth. "That's all you want to do." There was sudden change from the ponderous infantry of theory to the light and speedy infantry of practice. The regiment, relieved of a burden, received a new impetus. But there was much loss of valuable knapsacks, and, on the whole, very good shirts. But the regiment was not yet veteranlike in appearance. Veteran regiments in the army were likely to be very small aggregations of men. Once, when the command had first come to the field, some perambulating veterans, noting the length of their column, had accosted them thus: "Hey, fellers, what brigade is that?" And when the men had replied that they formed a regiment and not a brigade, the older soldiers had laughed, and said, "O Gawd!" Also, there was too great a similarity in the hats. The hats of a regiment should properly represent the history of headgear for a period of years. And, moreover, there were no letters of faded gold speaking from the colors. They were new and beautiful, and the color bearer habitually oiled the pole. Presently the army again sat down to think. The odor of the peaceful pines was in the men's nostrils. The sound of monotonous axe blows rang through the forest, and the insects, nodding upon their perches, crooned like old women. The youth returned to his theory of a blue demonstration. One gray dawn, however, he was kicked in the leg by the tall soldier, and then, before he was entirely awake, he found himself running down a wood road in the midst of men who were panting from the first effects of speed. His canteen banged rhythmically upon his thigh, and his haversack bobbed softly. His musket bounced a trifle from his shoulder at each stride and made his cap feel uncertain upon his head. He could hear the men whisper jerky sentences: "Say--what's all this--about?" "What th' thunder--we--skedaddlin' this way fer?" "Billie--keep off m' feet. Yeh run--like a cow." And the loud soldier's shrill voice could be heard: "What th' devil they in sich a hurry for?" The youth thought the damp fog of early morning moved from the rush of a great body of troops. From the distance came a sudden spatter of firing. He was bewildered. As he ran with his comrades he strenuously tried to think, but all he knew was that if he fell down those coming behind would tread upon him. All his faculties seemed to be needed to guide him over and past obstructions. He felt carried along by a mob. The sun spread disclosing rays, and, one by one, regiments burst into view like armed men just born of the earth. The youth perceived that the time had come. He was about to be measured. For a moment he felt in the face of his great trial like a babe, and the flesh over his heart seemed very thin. He seized time to look about him calculatingly. But he instantly saw that it would be impossible for him to escape from the regiment. It inclosed him. And there were iron laws of tradition and law on four sides. He was in a moving box. As he perceived this fact it occurred to him that he had never wished to come to the war. He had not enlisted of his free will. He had been dragged by the merciless government. And now they were taking him out to be slaughtered. The regiment slid down a bank and wallowed across a little stream. The mournful current moved slowly on, and from the water, shaded black, some white bubble eyes looked at the men. As they climbed the hill on the farther side artillery began to boom. Here the youth forgot many things as he felt a sudden impulse of curiosity. He scrambled up the bank with a speed that could not be exceeded by a bloodthirsty man. He expected a battle scene. There were some little fields girted and squeezed by a forest. Spread over the grass and in among the tree trunks, he could see knots and waving lines of skirmishers who were running hither and thither and firing at the landscape. A dark battle line lay upon a sunstruck clearing that gleamed orange color. A flag fluttered. Other regiments floundered up the bank. The brigade was formed in line of battle, and after a pause started slowly through the woods in the rear of the receding skirmishers, who were continually melting into the scene to appear again farther on. They were always busy as bees, deeply absorbed in their little combats. The youth tried to observe everything. He did not use care to avoid trees and branches, and his forgotten feet were constantly knocking against stones or getting entangled in briers. He was aware that these battalions with their commotions were woven red and startling into the gentle fabric of softened greens and browns. It looked to be a wrong place for a battle field. The skirmishers in advance fascinated him. Their shots into thickets and at distant and prominent trees spoke to him of tragedies--hidden, mysterious, solemn. Once the line encountered the body of a dead soldier. He lay upon his back staring at the sky. He was dressed in an awkward suit of yellowish brown. The youth could see that the soles of his shoes had been worn to the thinness of writing paper, and from a great rent in one the dead foot projected piteously. And it was as if fate had betrayed the soldier. In death it exposed to his enemies that poverty which in life he had perhaps concealed from his friends. The ranks opened covertly to avoid the corpse. The invulnerable dead man forced a way for himself. The youth looked keenly at the ashen face. The wind raised the tawny beard. It moved as if a hand were stroking it. He vaguely desired to walk around and around the body and stare; the impulse of the living to try to read in dead eyes the answer to the Question. During the march the ardor which the youth had acquired when out of view of the field rapidly faded to nothing. His curiosity was quite easily satisfied. If an intense scene had caught him with its wild swing as he came to the top of the bank, he might have gone roaring on. This advance upon Nature was too calm. He had opportunity to reflect. He had time in which to wonder about himself and to attempt to probe his sensations. Absurd ideas took hold upon him. He thought that he did not relish the landscape. It threatened him. A coldness swept over his back, and it is true that his trousers felt to him that they were no fit for his legs at all. A house standing placidly in distant fields had to him an ominous look. The shadows of the woods were formidable. He was certain that in this vista there lurked fierce-eyed hosts. The swift thought came to him that the generals did not know what they were about. It was all a trap. Suddenly those close forests would bristle with rifle barrels. Ironlike brigades would appear in the rear. They were all going to be sacrificed. The generals were stupids. The enemy would presently swallow the whole command. He glared about him, expecting to see the stealthy approach of his death. He thought that he must break from the ranks and harangue his comrades. They must not all be killed like pigs; and he was sure it would come to pass unless they were informed of these dangers. The generals were idiots to send them marching into a regular pen. There was but one pair of eyes in the corps. He would step forth and make a speech. Shrill and passionate words came to his lips. The line, broken into moving fragments by the ground, went calmly on through fields and woods. The youth looked at the men nearest him, and saw, for the most part, expressions of deep interest, as if they were investigating something that had fascinated them. One or two stepped with overvaliant airs as if they were already plunged into war. Others walked as upon thin ice. The greater part of the untested men appeared quiet and absorbed. They were going to look at war, the red animal--war, the blood-swollen god. And they were deeply engrossed in this march. As he looked the youth gripped his outcry at his throat. He saw that even if the men were tottering with fear they would laugh at his warning. They would jeer him, and, if practicable, pelt him with missiles. Admitting that he might be wrong, a frenzied declamation of the kind would turn him into a worm. He assumed, then, the demeanor of one who knows that he is doomed alone to unwritten responsibilities. He lagged, with tragic glances at the sky. He was surprised presently by the young lieutenant of his company, who began heartily to beat him with a sword, calling out in a loud and insolent voice: "Come, young man, get up into ranks there. No skulking'll do here." He mended his pace with suitable haste. And he hated the lieutenant, who had no appreciation of fine minds. He was a mere brute. After a time the brigade was halted in the cathedral light of a forest. The busy skirmishers were still popping. Through the aisles of the wood could be seen the floating smoke from their rifles. Sometimes it went up in little balls, white and compact. During this halt many men in the regiment began erecting tiny hills in front of them. They used stones, sticks, earth, and anything they thought might turn a bullet. Some built comparatively large ones, while others seemed content with little ones. This procedure caused a discussion among the men. Some wished to fight like duelists, believing it to be correct to stand erect and be, from their feet to their foreheads, a mark. They said they scorned the devices of the cautious. But the others scoffed in reply, and pointed to the veterans on the flanks who were digging at the ground like terriers. In a short time there was quite a barricade along the regimental fronts. Directly, however, they were ordered to withdraw from that place. This astounded the youth. He forgot his stewing over the advance movement. "Well, then, what did they march us out here for?" he demanded of the tall soldier. The latter with calm faith began a heavy explanation, although he had been compelled to leave a little protection of stones and dirt to which he had devoted much care and skill. When the regiment was aligned in another position each man's regard for his safety caused another line of small intrenchments. They ate their noon meal behind a third one. They were moved from this one also. They were marched from place to place with apparent aimlessness. The youth had been taught that a man became another thing in a battle. He saw his salvation in such a change. Hence this waiting was an ordeal to him. He was in a fever of impatience. He considered that there was denoted a lack of purpose on the part of the generals. He began to complain to the tall soldier. "I can't stand this much longer," he cried. "I don't see what good it does to make us wear out our legs for nothin'." He wished to return to camp, knowing that this affair was a blue demonstration; or else to go into a battle and discover that he had been a fool in his doubts, and was, in truth, a man of traditional courage. The strain of present circumstances he felt to be intolerable. The philosophical tall soldier measured a sandwich of cracker and pork and swallowed it in a nonchalant manner. "Oh, I suppose we must go reconnoitering around the country jest to keep 'em from getting too close, or to develop 'em, or something." "Huh!" said the loud soldier. "Well," cried the youth, still fidgeting, "I'd rather do anything'most than go tramping 'round the country all day doing no good to nobody and jest tiring ourselves out." "So would I," said the loud soldier. "It ain't right. I tell you if anybody with any sense was a-runnin' this army it--" "Oh, shut up!" roared the tall private. "You little fool. You little damn' cuss. You ain't had that there coat and them pants on for six months, and yet you talk as if--" "Well, I wanta do some fighting anyway," interrupted the other. "I didn't come here to walk. I could 'ave walked to home--'round an' 'round the barn, if I jest wanted to walk." The tall one, red-faced, swallowed another sandwich as if taking poison in despair. But gradually, as he chewed, his face became again quiet and contented. He could not rage in fierce argument in the presence of such sandwiches. During his meals he always wore an air of blissful contemplation of the food he had swallowed. His spirit seemed then to be communing with the viands. He accepted new environment and circumstance with great coolness, eating from his haversack at every opportunity. On the march he went along with the stride of a hunter, objecting to neither gait nor distance. And he had not raised his voice when he had been ordered away from three little protective piles of earth and stone, each of which had been an engineering feat worthy of being made sacred to the name of his grandmother. In the afternoon the regiment went out over the same ground it had taken in the morning. The landscape then ceased to threaten the youth. He had been close to it and become familiar with it. When, however, they began to pass into a new region, his old fears of stupidity and incompetence reassailed him, but this time he doggedly let them babble. He was occupied with his problem, and in his desperation he concluded that the stupidity did not greatly matter. Once he thought he had concluded that it would be better to get killed directly and end his troubles. Regarding death thus out of the corner of his eye, he conceived it to be nothing but rest, and he was filled with a momentary astonishment that he should have made an extraordinary commotion over the mere matter of getting killed. He would die; he would go to some place where he would be understood. It was useless to expect appreciation of his profound and fine senses from such men as the lieutenant. He must look to the grave for comprehension. The skirmish fire increased to a long chattering sound. With it was mingled far-away cheering. A battery spoke. Directly the youth would see the skirmishers running. They were pursued by the sound of musketry fire. After a time the hot, dangerous flashes of the rifles were visible. Smoke clouds went slowly and insolently across the fields like observant phantoms. The din became crescendo, like the roar of an oncoming train. A brigade ahead of them and on the right went into action with a rending roar. It was as if it had exploded. And thereafter it lay stretched in the distance behind a long gray wall, that one was obliged to look twice at to make sure that it was smoke. The youth, forgetting his neat plan of getting killed, gazed spell bound. His eyes grew wide and busy with the action of the scene. His mouth was a little ways open. Of a sudden he felt a heavy and sad hand laid upon his shoulder. Awakening from his trance of observation he turned and beheld the loud soldier. "It's my first and last battle, old boy," said the latter, with intense gloom. He was quite pale and his girlish lip was trembling. "Eh?" murmured the youth in great astonishment. "It's my first and last battle, old boy," continued the loud soldier. "Something tells me--" "What?" "I'm a gone coon this first time and--and I w-want you to take these here things--to--my--folks." He ended in a quavering sob of pity for himself. He handed the youth a little packet done up in a yellow envelope. "Why, what the devil--" began the youth again. But the other gave him a glance as from the depths of a tomb, and raised his limp hand in a prophetic manner and turned away. | Summary: The regiment rests during the second day of their march, and that night, they cross a bridge and sleep again. On the morning of the third day, they again move out and march to a forest. They remain there for several days. The regiment rests during the second day of their march, and that night, they cross a bridge and sleep again. On the morning of the third day, they again move out and march to a forest. They remain there for several days. On "one gray dawn" the whole regiment begins to run as if running toward a battle, but there is no battle. The regiment walks and then halts, and the soldiers continue to move from place to place. There is much grumbling among the men because of the constant walking and stopping. On occasion, the regiment sees skirmishers in the distance and hears the sounds of battle. The regiment comes upon a dead soldier, and Henry tries "to read in dead eyes the answer to the Question." Henry continues to challenge, internally, the intelligence of the generals who are directing the troop movements, and he feels hatred toward the lieutenant who enforces troop discipline by keeping him marching in rank. Henry considers that if he were to die quickly, he could end his anguish. The regiment comes upon a battle in the distance, and the men begin to prepare for battle. As the chapter ends, the loud soldier tells Henry that he expects to die in battle, and he hands Henry a packet which he asks Henry to take to his family. | 4,145 | 338 | booksum | en |
Write a title and summarize: The segmentation of the vertebrate body is laid down during early embryogenesis. The formation of signaling gradients, the periodic expression of genes of the Notch-, Fgf- and Wnt-pathways and their interplay in the unsegmented presomitic mesoderm (PSM) precedes the rhythmic budding of nascent somites at its anterior end, which later develops into epithelialized structures, the somites. Although many in silico models describing partial aspects of somitogenesis already exist, simulations of a complete causal chain from gene expression in the growth zone via the interaction of multiple cells to segmentation are rare. Here, we present an enhanced gene regulatory network (GRN) for mice in a simulation program that models the growing PSM by many virtual cells and integrates WNT3A and FGF8 gradient formation, periodic gene expression and Delta/Notch signaling. Assuming Hes7 as core of the somitogenesis clock and LFNG as modulator, we postulate a negative feedback of HES7 on Dll1 leading to an oscillating Dll1 expression as seen in vivo. Furthermore, we are able to simulate the experimentally observed wave of activated NOTCH (NICD) as a result of the interactions in the GRN. We esteem our model as robust for a wide range of parameter values with the Hes7 mRNA and protein decays exerting a strong influence on the core oscillator. Moreover, our model predicts interference between Hes1 and HES7 oscillators when their intrinsic frequencies differ. In conclusion, we have built a comprehensive model of somitogenesis with HES7 as core oscillator that is able to reproduce many experimentally observed data in mice. Somitogenesis is an embryonic process that provides the basis for the mesodermal segmentation of the vertebrate body. Somites are derivatives of the presomitic mesoderm (PSM), a mesenchymal tissue that is formed during gastrulation and maintained by proliferation of cells in the tail bud. They are epithelial balls of cells that separate from the anterior end of the PSM to both sides of the neural tube. In mice, approximately every two hours one pair of somites is formed until proliferation in the tail bud stops and a species-specific number of somite pairs has been generated [1]. Fundamental to somitogenesis is the formation of a segmental boundary between the last formed somite and the unsegmented PSM. Before a boundary becomes morphologically visible, wave-like gene expression patterns propagate from the posterior to the anterior end of the PSM with the same periodicity as somites are formed [2]. Most prominent among these cycling genes are those involved in the Delta/Notch (D/N) pathway, like Lfng and the helix-loop-helix transcription factors Hes1, Hes5, Hes7 and Heyl. They are induced by the NOTCH intracellular domain (NICD), which is cleaved off from the NOTCH receptor upon binding to DELTA or JAGGED ligands at adjacent cells and acts subsequently as co-transcription factor in the nucleus of NOTCH expressing cells [3]. NICD shows a cycling and wave-like expression in the PSM [4]. D/N signaling and Hes7 oscillation are essential for somitogenesis [5], [6]. For example, loss of NOTCH1 function resulted in delayed and disorganized somitogenesis [7]. Similarly, in mice lacking the NOTCH ligand DELTA-LIKE 1 (DLL1) or the down-stream effector HES7 somites are not properly segmented and display a disrupted rostral-caudal polarity [8], [9]. In contrast, oscillating expression of Lfng in the posterior PSM seems to be dispensable for the formation of somites that later give rise to sacral and tail vertebrae [10], [11]. Other genes required for normal somitogenesis belong to the Fgf and Wnt/β-catenin signaling pathways. Both Fgf8 and Wnt3a are transcribed in the growth zone of the tail bud but not in the more anterior region of the PSM. A slow decay of Fgf8 mRNA leads to a graded expression of FGF8 protein levels from the posterior to the anterior end of the PSM [12]. Likewise, a posterior to anterior gradient of nuclear β-catenin is observed [13]. A third gradient of retinoic acid (RA) is established in the reverse direction and thought to suppress Fgf8 expression [14]. Genes downstream of the Fgf pathway cycle in phase with respect to D/N oscillations, whereas genes belonging to Wnt/β-catenin signaling cycle in anti-phase [15]. Experimental manipulations of the Fgf or Wnt/β-catenin pathway also impair somite formation [13], [16], and inhibition of casein kinase 1, which is downstream of Wnt, lengthens the period of the somitogenesis clock [17]. In Mesp2 deficient embryos, somite boundary formation is lost [18]. MESP2 induces the expression of Epha4 [19]. In chick, the EPHA4 receptor binds to the ephrin B2 ligand on cells across the future boundary and thereby triggers furrow formation and cell epithelialization at the gap between the forming somite and the PSM [20]. Mesp2 is expressed periodically by joint binding of NICD and the T-box transcription factor TBX6 in its promoter region resulting in a narrowing stripe of Mesp2 expression at the anterior PSM [21]. While Tbx6 expression is static and restricted to the PSM and is rapidly down-regulated as the somites form, NICD expression is dynamic, forming a wave moving in anterior direction through the PSM and contracting antero-posteriorly in width as it nears the anterior end [4]. Based on the promoter information for Mesp2 and additional evidence that FGF8 suppresses posterior Mesp2 expression, Oginuma et al. formulated a model that describes how dynamic NICD induces Mesp2 expression patterns in the PSM [22]. Later, they simulated Mesp2 and TBX6 expression with a system of differential equations in a computer model of a one-dimensional array of cells [23]. Other models employ a modulo function on an Fgf gradient [24] to generate the NICD wave, or a Boolean variable for NICD, which is repressed by the action of a Hes7 oscillator with an empirically adjusted oscillation period [25]. We aim to develop an integrated model that depicts the causal chain leading from processes in the growth zone – unfortunately still incompletely known – to the dynamic gene expression patterns in the PSM wave zone and to segmentation of the PSM into somites. In particular, we are driven by the following questions: These questions are connected to our central question: what is the core oscillator driving somitogenesis and how does it work? While this manuscript was in the review process, Hester et al. published such an integrated model for chick somitogenesis, in which repression of D/N signaling by LFNG serves as the core oscillator [26]. Here, we propose an extended theoretical gene regulatory network (GRN) for Mesp2 oscillation that is based on a Hes7 feedback oscillator [27] driven by dynamic NICD expression with LFNG as modulator/enhancer of D/N signaling. Additionally, we assume a negative feedback of HES7 on Dll1 expression and hypothesize an influence of the Wnt3a signaling gradient on the decay of NICD. Figure 1 shows a schematic representation of the vertebrate segmentation process and the underlying gene/protein expression patterns that we consider to be essential. By incorporating the extended GRN in a multi-cell simulation program of the growing PSM that allows real-time observation of gene expression in thousands of virtual cells [28], we are able to model the dynamics of NICD expression and link D/N signaling to the Mesp2/Ephrin system. As result we observe dynamic wave-like expression of NICD and Dll1 in silico as seen in vivo [4], [29] as well as periodic Mesp2 expression along the growing PSM [4]. Introducing a gene “Epha4” downstream of MESP2 we obtain regular formation of “Epha4” expression maxima. The usefulness of our in silico system is demonstrated by the elimination of Hes7 or Ripply2 from the GRN, which results in non-oscillatory NICD and Mesp2 expression patterns moving from anterior to posterior or, in the case of absent RIPPLY2, in a double-striped Mesp2 expression as observed similarly in respective mouse models [6], [30]–[32]. Furthermore, we show the occurrence of beat [33] in oscillating gene expression resulting from the interference of two genetic oscillators with different eigenfrequencies. A lot of experimental information in biology resides in pictures derived from experiments showing their results by in situ staining. These results lead to hypotheses formulated e. g. in the form of GRNs. In the field of somitogenesis, Gonzalez et al. [34] has built a database of all relevant experiments and examined whether the GRNs discussed so far can explain the observed results. They found gaps in our understanding and tried to fill them with hypothetical interactions. Still missing is a computational validation whether the postulated GRN can really explain observed gene expression patterns. Therefore, one would need a gene- and cell-based model of the process in question. Our model is intended as a first step in this direction. Somitogenesis is comprised of several subprocesses such as the growth of the PSM, oscillatory gene expression, synchronization of the oscillators, boundary formation between PSM and the next forming somite, somite polarization, and somite epithelialization. Several mathematical models exist for some of the processes, which provide a basic understanding of the described phenomena. However, each model has its own assumptions and simplifications, so it is not clear whether existing partial models are ‘consistent and integrable with one another’ [26]. Furthermore, there are experimentally generated phenotypes, which can be fully understood only by the interaction of several parts of the system, each of them modeled separately until now (for an example, see our in silico Hes7 knock-out experiment below.) We intended to build a comprehensive model of somitogenesis, in which most processes generated by the action of a GRN i. e. by integrating the differential equations describing the processes. However, the proliferation in the growth zone and the deactivation of Fgf8 and Wnt3a expression when cells leave the growth zone were put in ‘by hand’ and are controlled by the program. We introduced EPH4A as a marker, which has the only task to trigger the shape of a cell to indicate boundary formation at the anterior PSM. This process is effected by the program when a certain threshold of EPH4A protein concentration is reached in the simulation by the GRN. We designed our program with the intention that a user can easily change the numerical values of the rate constants, the Hill functions, or take out genes. The resulting expression pattern of a chosen gene product can be followed in real time. We therefore attached a detailed graphical user interface (GUI) to our program (see the screenshots in the user manual provided as supplementary Text S1.) To model gene expressions in the PSM we use essentially the same methodology as recently described in Tiedemann et al. 2007 [28], i. e. a gene- and cell-based simulation program that numerically solves differential equations describing a gene regulatory network in each cell and displays the actual concentration of a selected gene product by color intensity (virtual in situ staining). The following improvements were made to the program: A growth zone of several cell layers now extends the rectangular geometry of the PSM. During growth of the PSM one cell of each column along the growth direction is randomly chosen for mitosis. Thereby the program allocates a new instance of a Java ‘cell’ object, which inherits all concentration values of its mother cell. A new ‘cell’ is created at the location of its ‘mother cell’ and then pushes stepwise all other cells of the respective column towards the posterior end of the PSM. The corresponding movements and position changes are computed by the program. The growth zone of the PSM is defined by the program to encompass the last n layers (user defined, default value: n = 15), i. e. planes perpendicular to the growth direction. If a cell leaves the growth zone Fgf8 and Wnt3a expression is shut down by the program, as we have no GRN modeling this process. The diffusion of FGF8 and WNT3A protein is not simulated in detail since we assume the gradients to be mostly determined by the intracellular decay of the respective mRNAs. So we assume a very short diffusion range for FGF8 and WNT3A [35], i. e. each cell receives only the proteins from its nearest neighbors. The concentrations are averaged and act immediately on the targets of the respective signal transduction pathway. This means, we assume the intervening processes are fast compared to the mRNA decay, which determines the dynamics and scale of the gradients. Furthermore, we introduce distinct variables for cellular and nuclear concentrations of proteins and the respective mRNAs. The distinction in compartments is made for the oscillatory factors HES1/7, MESP2, NICD and LFNG, but not for the slow-changing concentrations of proteins and mRNAs of Notch1, Tbx6, Fgf8 and Wnt3a. For the DLL1 ligand and the NOTCH receptor we set separate variables in the cytoplasm and membrane compartments. We validated our model system by the in silico elimination of Hes7, which results in a constant, receding stripe of Mesp2 expression moving in anterior to posterior direction with the growing PSM (Figure 4, Video S6). This result is consistent with experimentally observed data in mice deficient for Hes7 [6], [25]. A constantly anterior to posterior moving Mesp2 expression in the PSM can be also observed when the influence of HES7 on the Dll1 promoter is eliminated. Although Hes7 expression is oscillatory it shows no discernible wave in the PSM, because constant DLL1 and NOTCH1 expression result in constant production of NICD. So Mesp2 is consequently moving within the borders set by TBX6 and FGF8 expression in the growing PSM (data not shown). A similar Mesp2 expression pattern was observed in vivo in embryos expressing NICD throughout the PSM [30]. We introduced a term for constant cytoplasmic NICD production in our simulation and observed again a constant, receding stripe of Mesp2 expression moving in anterior to posterior direction (data not shown). When we eliminated Ripply2 from the GRN we observed two stripes of Mesp2 expression (Figure 5). Similar expression patterns for Mesp2 were observed in mice deficient for Ripply2 [31], [32]. By the usage of the chemical compound SU5402 to inhibit Fgf signaling, Niwa et al. observed a broadened stripe of Mesp2 as result of a precociously expression in the next clock cycle [25]. To simulate an inhibition of Fgf signaling we reduced the FGF8 protein production rate during a simulation run by 50% after 600 minutes and observed a similar expanded Mesp2 expression (Figure 6, Video S7). Genetic oscillators of the Hes1/7 type are understood to result from a negative feedback with delays. These delays can be incorporated into a mathematical description in various ways. Either directly as delayed time arguments describing the duration of transcription, translation and transport processes [39], [65], resulting in delay-differential-equations, or, alternatively, as chains of transport equations [64], possibly enhanced by the introduction of nonlinearities like Michaelis-Menten or Hill-type functions [42] describing, for example, saturated decay processes. A third possibility is the explicit modeling of intracellular diffusion of proteins and mRNA in the cytoplasm of a cell [66]. Although delay models require only two equations per gene and the delays, for instance caused by splicing of Hes7 introns [65], are easier to measure than in our compartment model, we think that delay equations used so far have two drawbacks. First, within delays many steps in gene-expression processes are hidden. As probably most processes like splicing, transport between nucleus and cytoplasm, protein and mRNA decay in the eukaryotic cell are controlled by signal transduction pathways, several modes of cross-talk [67] are neglected. Of course, it is not meaningful to model each step in transcription and translation by one differential equation. The transport steps and the saturated decay implemented in our model should be understood as representative for all these cellular processes. So, a cross-over model restricting the delays to transcription and translation would probably be the best model for future simulations. Second, our model uses nonlinearities and saturation functions to consider proteasome-effected degradation of transcription factors. Proteasomes are located in the cytoplasm as well as in the nucleus [68], [69]. We introduce nonlinearities to allow a possible saturation of the proteasome machinery in the nucleus but neglect this possibility in the cytoplasm. However, if one simulates explicitly phosphorylation and ubiquitination of a protein before its destruction in the proteasome [70], one could assume saturated decay also in the cytoplasm [43]. For further information concerning different processes involving NICD in the nucleus and cytoplasm see [71], [72]. Because degradation processes in an eukaryotic cell are affected by protein complexes, i. e. molecular machines like proteasomes for protein disposal or exosomes for mRNA destruction, nonlinear descriptions could be appropriate for other processes as well. Every machine has a saturation threshold that can be overwhelmed by substrate molecules when their concentration is too high. Another important issue, which needs more effort, is the role of transcription cofactors and their influence on the Hill coefficients of associated transcription factors. The higher the Hill coefficient and the degree of cooperation, the higher is the propensity to oscillations [42]. Many genes were found to oscillate in the PSM of mouse embryos [15], most of them are downstream of the Fgf, Wnt and D/N –pathways. Genes downstream of the Fgf pathway cycle with respect to D/N oscillations, whereas genes belonging to Wnt signaling cycle in anti-phase. Some of the downstream genes in both pathways act as inhibitors along the signal transduction cascade, forming negative feedback loops and allow for oscillations. Two scenarios are conceivable: an oscillator in one pathway acts as master clock and controls the others, or alternatively, no master clock exists and all cycling genes are equally important in the oscillatory network. A model for the latter case was developed by Goldbeter and Pourquie [44]. Genes of all three pathways generate three oscillators, which are coupled and synchronized by genetic interactions between them. We will argue for the first scenario with the D/N pathway as the central driver. First, there is evolutionary conservation. Comparing cycling gene expression in mouse, chick, and zebrafish, Krol et al. [73] found that ‘conservation of cyclic genes for all three species is limited to orthologs of the HES/her transcriptional repressors’. For example, comparing the Wnt pathway between chick and mouse only Axin2 oscillates in both species [73], and in anolis lizards, only the orthologs of Hes1, Hes7, Dll1, and Dll3 are dynamic whereas other genes like Lfng are not oscillating [74]. Second, several experimental data point to the same direction. In mice, most of the FGF8 targets, which oscillate in phase with D/N cycling genes, are controlled either directly by NICD as co-transcription factor, as in the case of Dusp6 and Spry4, or indirectly via HES7 like Spry2 and Dusp4 [6], [36], [75]. Moreover, if one considers also the activation of Snail and Nrarp by NICD [76], [77], it seems reasonable to suppose that D/N signaling controls all genes that cycle in phase to D/N oscillations [15]. D/N signaling controls also Nkd1, which interact with components of the Wnt pathway [78]. One of the cycling genes in this pathway is Axin2, which have a negative feedback on Wnt signaling and could be a critical component of a Wnt oscillator [79]. However, it was shown that Axin2 deficient mice show no somitogenesis phenotype [80]. Of course, all these arguments do not prove conclusively that D/N is the central oscillator in mice. However, it supports the assumptions made in our model, which sets D/N signaling at the center. The complex D/N oscillator consists of two interlocking negative feedback loops. On the one hand, Lfng, which is dynamically expressed in the PSM, modulates D/N signaling by glycosylation of the NOTCH receptor. It is induced by NICD and suppressed by HES7 and was therefore considered to be a core molecular mechanism of the somitogenesis clock [81], [82]. On the other hand, the negative feedback of Hes7, which is driven by NICD and FGF8, onto itself, represses also Lfng expression. There are in silico models that take the Lfng negative feedback loop as the core of the somitogenesis clock [26], [44], while others emphasize the role of Hes7 [25]. Niwa et al., for example, assumed a direct suppression by HES7 on NICD expression [25]. However, both models cannot explain, in our opinion, some crucial experiments. LFNG protein is secreted from a cell to terminate its action in the secreting cell but probably not as mechanism for cell-cell communication [83]. The ‘Lfng only’ models cannot describe the uniformly receding Mesp2 expression observed in the PSM of Hes7 knock-out embryos [6], [25]. These models have to postulate a direct influence of NICD on Dll1 expression in the same cell, otherwise an oscillation in one cell could not be transmitted to neighboring cells, i. e. there would be no information transfer between cells, which is needed for the postulated role of D/N signaling in synchronizing cellular oscillators of the PSM [84]. Furthermore, Lfng expression seems not to be required for proper somitogenesis in the late tail bud phase [10], [11], and Hes7 expression is still dynamic in Lfng deficient mice [6]. In addition, Lfng shows a constant expression in zebrafish and Medaka [85]. Contrary to these findings, Oginuma et al. recently reported a requirement for oscillating Lfng expression in the posterior PSM for proper somitogenesis [23], and Niwa et al. observed a damping in the amplitude of HES7 oscillations in Lfng deficient compared to wild type mice [25]. A model that postulates a direct interaction of HES7 on NICD has difficulties to explain the fact that Dll1 was found to be cycling in the PSM [29]. One would have to assume a direct interaction of NICD on Dll1 or a longer causal chain, in which, for example, NICD controls Nkd1, which controls Wnt signaling that controls Dll1. Here, we show that a simple model analogous to the zebrafish network is able to describe the dynamic NICD expression in the PSM as it was shown by Morimoto et al. [4]. Retinoic acid (RA) is important for synchronizing and balancing the development of left and right halves of the PSM [98]. RA is expressed in the somites and forms an anterior to posterior gradient opposing the posterior to anterior gradient of Fgf8. The opposing action of both gradients was modeled by Goldbeter et al. [99] and results in a sharp expression cut-off at the anterior boundary of the FGF8 gradient. Because there is not enough information about the control of RA in somites, which would allow us to incorporate RA signaling in our GRN, it is currently not included in our simulations. However, we modeled an inhibitory action of FGF8 on Mesp2 expression with a high hill coefficient to ensure a steep drop-off at the anterior end of FGF8 expression. Apart from these considerations, our model is intended to model somitogenesis in the tail bud phase. Cunningham et al. [100] have shown that during mouse embryogenesis from E9. 5 to E13. 5 Mesp2 expression and somitogenesis was not changed in Raldh2 knock-out mice, which are unable to produce RA in the somites. They concluded ‘that as early as E9. 5 Raldh2 is not required to limit the anterior extent of the caudal Fgf8 expression zone. ’ While it is known, for example, that Dusp6 is controlled by FGF8-ERK1/2 via the Ets family of transcription factors [101], it is yet unknown which of the FGF8 downstream factors activate Hes7 and inhibit Mesp2 [22], [36]. Once this is known, we could extend our model by more detailed modeling of Fgf8 and Wnt3a signaling and the corresponding downstream genes, some of which show also cycling expression [15] due to the various negative feedbacks that are discussed for these signal transduction pathways [102]. For a single-cell-simulation this was done by Goldbeter and Pourquie [44], and for a 2-D simulation of chicken somitogenesis by Hester et al. [26]. Recently, Niwa et al. [25] observed that the expression of pERK and DUSP4, which are downstream of FGF8, is not constantly receding but periodically covers and frees the MESP2 expressing region of the PSM. This could be a result of the negative feedback of DUSP4, driven by HES7 oscillations, on Fgf8 signaling and be important for the control of Mesp2 expression in the future somite [25]. Furthermore, besides refining the Mesp2, Ripply2, and Tbx6 expression patterns by incorporating the mechanisms proposed by Takahashi et al. [24] a much more detailed modeling of the Ephrin/Eph receptor system with forward as well as backward signaling components [103] should be considered, as also other genes in this network cycle or are controlled by HES7 [36], [73]. In our simulations, all cells start synchronously in the growth zone and remain synchronized because daughter cells inherit the oscillation phase of their mother cells. So, synchronization of cellular oscillators by D/N coupling is not needed. We achieved a partial solution of the synchronization problem in an earlier version of our model with a growth zone comprising only one single cell layer. When we coupled the HES1 transcription factor as inhibitor to the Dll1 promoter while Notch signaling activates Hes1, we observed similar synchronized cellular oscillations in neighboring cells as for a delay differentiation model of zebrafish [39], [86] or the Hes7 oscillator of mouse [58]. However, we achieved synchronization only when we consider the binding of NICD as dimer [38] and when the relative phases of Hes oscillations in all cells generated in the one cell-layered growth zone differ not more than 25% (Figure S9). Uriu et al. have demonstrated that one has to include random cell movements into the PSM to achieve faster synchronization [104]. Such a random cell motility gradient downstream of FGF8 was recently described for chick embryos [105]. Furthermore, the assumption of D/N signaling to function exclusively through nearest neighbor communication could be reconsidered. In Drosophila, for example, dynamic filopodia transmit D/N signals during bristle formation [106]. Similar cellular extensions bearing Delta ligands were observed in zebrafish [107]. An even more radical deviation from the common view on D/N signaling might be considered by the finding that DLL4 incorporated in endothelial exosomes might be transferred and integrated into the cell membrane of distant cells [108]. Somitogenesis describes not only border formation, but also comprises the mesenchymal to epithelial transition in the outer layer of cells of the forming somite. Snail and Zeb2 are genes that are well known to be involved in epithelial-to-mesenchymal transition (EMT) by controlling E-cadherin [109] but also show cyclic expression in the PSM [29], [76]. Finally, somitogenesis requires an extra-cellular matrix (ECM) of fibronectin surrounding the PSM [110]. Jülich et al. demonstrated that in zebrafish reverse signaling by EphrinB2a is sufficient to initiate Itgα5 clustering, alleviates non-cell-autonomous transinhibition, which prevents fibronectin activation within the PSM, but induces fibronectin matrix assembly along somite borders [111]. Taken together, these findings reinforce the need to include ECM and integrin signaling into the modeling of somitogenesis. However, the translation of gene expression patterns into the control of cell adhesion and cell shape, as well as the interaction with the extracellular matrix would require the development of a model for the cell skeleton and other features, which should not only be realistic enough but also computable in a simulation with thousands of cells. The influence of the NICD wave on the dynamics of Mesp2 and Ripply2 expression, activated by TBX6 and repressed posteriorly by FGF8, was recently simulated in several mathematical models. To reproduce NICD expression as experimentally observed the wave was generated either by a wave function [23], by a modulo function on an Fgf gradient [24], or by a Boolean variable for NICD which is repressed by the action of a Hes7 oscillator with an empirically adjusted oscillation period [25]. Here, like in Hester et al. [26], we propose a model in which the NICD wave in the PSM results from a GRN. However, in our model of the mouse core oscillator for somitogenesis a negative feedback of the HES7 oscillator on the expression of Dll1 leads to periodic expression of NICD, while Lfng is considered not as central part of the core oscillator but as a modulator of the Hes7-D/N oscillator. Its oscillation slows down because the degradation of NICD is influenced by a Wnt3a gradient in the posterior PSM. Since quantitative information on rate constants for production and degradation of mRNAs and proteins is mostly missing, we classify the genes in our model with fast or slow dynamics. However, our model is able to reproduce a great deal of experimentally observed data in mice. Encouraged by the agreement between experiment and our simulation, we hypothesize that HES7 binds to the Dll1 promoter. To prove this hypothesis experimentalists could perform in vivo chromatin immunoprecipitation (ChIP) analyses according to a protocol previously described by Bessho et al. 2003 [5]. In the following, we will summarize the achievements but also the deficiencies of our model in bullet-point form: Achievements Deficiencies However, our model can answer at least theoretically some of the questions listed in a recent review [79] concerning the nature of the somitogenesis oscillator and its interaction with the Wnt3a gradient. Furthermore, we now have a model for the generation of dynamic NICD expression in the PSM, which is also robust to our parameter variations. In addition, we can connect our model to the model of somite border formation as formulated by Oginuma et al. [22], which can explain important aspects of somitogenesis. Furthermore, our programming framework makes it easy to expand the model in future works. Finally, we are pleading for Hes7 as the central pacemaker of the somitogenesis clock with Lfng having only a modulatory role. This scenario can explain some observations other models cannot, and is in accordance with the evolutionary conservation of the somitogenesis clock. This plea is made not only by arguments, but also by demonstrating its viability in computer simulations. However, in the end the in vivo experiment has to decide whether HES7 binds to the Dll1 promoter – as our model assumes – or not. The GRN is represented by 38 differential equations. The equations, the specific rate constants for the decay, production, as well as import of proteins and mRNAs for each gene are given in the supporting information (Table S1). The program is written in Java with the 3D-extension. For rendering the concentrations of the different gene products their values have to be normalized to lie in a range between zero and one. The differential equations are numerically solved with a fourth order Runge-Kutta-algorithm. Videos were made using camstudio software. The program writes all data of user selected cells to file. Plots were performed with the GNU-plot software. This is especially useful if one does not know beforehand the concentration range of a gene product that is selected to view in the simulation. The program can be downloaded at www. helmholtz-muenchen. de/en/ieg/downloads/simulation11. As the program requires the Java 3D API, which for legal reasons cannot be packaged into the downloadable file by us, users must ensure that it is installed on their computers before trying to run the program. The source code is available upon personal request. | Title: From Dynamic Expression Patterns to Boundary Formation in the Presomitic Mesoderm Summary: Somitogenesis is a process in embryonic development establishing the segmentation of the vertebrate body by the periodic separation of small balls of epithelialized cells called somites from a growing mesenchymal tissue, the presomitic mesoderm (PSM). The basic mechanisms are often discussed in terms of the clock-and-wave-front model, which was proposed already in 1976. Candidate genes for this model were found only in the last fifteen years with the cyclically expressed Hairy/Hes genes functioning as the clock and posteriorly expressed Fgf, Tbx6, and Wnt genes establishing the gradient (s). In addition, the Delta/Notch signal transduction pathway seems to be important for boundary formation between forming somites and the remaining PSM by inducing Mesp2 expression just behind a future somitic boundary. Although many in silico models describing partial aspects of somitogenesis already exist, there are still conflicts regarding the mechanisms of the somitogenesis clock. Furthermore, a simulation that fully integrates clock and gradient was only recently published for chicken. Here, we propose a cell- and gene-based computer model for mammalian somitogenesis, simulating a gene regulatory network combining clock (Hes1/7) and gradient (Tbx6, Fgf8, Wnt3a) with Delta/Notch signaling resulting in dynamic gene expression patterns as observed in vivo finally leading to boundary formation. | 7,728 | 334 | lay_plos | en |
Summarize: These 3,700-year-old jars were discovered in an ancient palatial wine cellar unearthed by researchers at Tel Kabri in July 2013. The team worked in day and night shifts to excavate a total of 40 intact vessels during its six-week dig. These 3,700-year-old jars were discovered in an ancient palatial wine cellar unearthed... Read More These 3,700-year-old jars were discovered in an ancient palatial wine cellar unearthed by researchers at Tel Kabri in July 2013. The team worked in day and night shifts to excavate a total of 40 intact vessels during its six-week dig. Close A 3,700-year-old wine cellar still holding vestiges of the drink has been unearthed in the Near East, potentially offering modern man a true taste of the past. The excavation in the ancient city of Tel Kabri, in Israel uncovered 40 jars in sizes that could have filled about 3,000 modern wine bottles. The residue suggested they once contained both white and red wine, made with additives that included juniper berries, cinnamon bark, mint and myrtle. The group had been digging since 2005 at the site, believed to be an ancient Canaanite palace and decorated with Aegean-style frescoes, the only such art to be found in Israel so far, according to a presentation today in Baltimore at the annual meeting of the American Schools of Oriental Research. “We’re thinking it’s a palatial wine cellar,” said Eric Cline, one of the archaeologists and the chairman of the Department of Classical and Near Eastern Languages and Civilizations at George Washington University. Two-thousand liters of wine isn’t enough for the general population. “It’s just enough for the ruler and his household.” Researchers located the cellar near the ruins of what was likely a banquet hall. The hall and cellar were destroyed in what may have been an earthquake. Photographer: Eric H. Cline, George Washington University George Washington University alumnus Zach Dunseth removes dirt and debris from ancient wine jars while excavating the ruins of a Canaanite city that dates back to approximately 1700 B.C. Close George Washington University alumnus Zach Dunseth removes dirt and debris from ancient... Read More Close Open Photographer: Eric H. Cline, George Washington University George Washington University alumnus Zach Dunseth removes dirt and debris from ancient wine jars while excavating the ruins of a Canaanite city that dates back to approximately 1700 B.C. Andrew Koh, an assistant professor of classical studies at Brandeis University, extracted the residue at the site and brought the samples to analyze at a lab at the Waltham, Massachusetts-based university. The consistency of the leftovers suggested the wine mix was made using a closely followed recipe. Trade Route The additives were probably there to preserve the wine, and some came from non-local plants, suggesting thriving trade routes. It was “a very luxurious drink reserved for special occasions,” said Assaf Yasur-Landau, the chairman of the department of maritime civilizations at the University of Haifa, and co-director of the excavation. The recipe was similar to medicinal wines used in ancient Egypt, the researchers said. “We can imagine it was like if you take a resinated wine and pour cough syrup in,” Yasur-Landau said. Researchers plan to continue analyzing the residue, in hopes of discovering enough information to recreate the flavor. The archaeologists had to work quickly to get the jugs out before winter, when the weather likely would destroy them. Working in two teams, they finished just before the scheduled end of the dig. There may be more wine containers yet to be found. A few days before the dig ended, the team discovered two doors leading out of the wine cellar, which may point toward additional storage rooms. A dig is scheduled for 2015 to explore further. To contact the reporter on this story: Elizabeth Lopatto in San Francisco at elopatto@bloomberg.net To contact the editor responsible for this story: Reg Gale at rgale5@bloomberg.net One of the world’s oldest and largest wine cellars has just been unearthed in Israel, according to a presentation today at the annual meeting of the American Schools of Oriental Research in Baltimore. The cellar dates to approximately 1700 B.C., and was found at a site called Tel Kabri within the ruins of a northern Canaanite city. Its age predates both the Bible and the Dead Sea Scrolls by hundreds of years. Climate Change Rewrites World Wine List The researchers christened a three-foot-long jar in the cellar “Bessie.” “We dug and dug, and all of a sudden, Bessie’s friends started appearing — five, 10, 15, ultimately 40 jars packed in a 15-by-25-foot storage room,” Eric Cline, chair of George Washington University’s Department of Classical and Near Eastern Languages and Civilizations, said in a press release. Cline, who co-directed the project, continued, “This is a hugely significant discovery — it’s a wine cellar that, to our knowledge, is largely unmatched in its age and size.” The 40 jars have a capacity of roughly 8453.5 cups, meaning the cellar could have held the equivalent of nearly 3,000 bottles of reds and whites. “The wine cellar was located near a hall where banquets took place, a place where the Kabri elite and possibly foreign guests consumed goat meat and wine,” said the other co-director Yasur-Landau, chair of the Department of Maritime Civilizations at the University of Haifa. “The wine cellar and the banquet hall were destroyed during the same violent event, perhaps an earthquake, which covered them with thick debris of mud bricks and plaster.” Video: What Ancient Wine Tasted Like Andrew Koh of Brandeis University also worked on the project. Using a technique called organic residue analysis, he studied some of the jar fragments. They yielded traces of tartaric and syringic acids, both key components in wine. Koh also detected other ingredients popular in ancient wine making (and modern day cocktails!), including honey, mint, cinnamon bark, juniper berries and resins. The Julia Child of wine during those times must have come from ancient Egypt, because the recipe is similar to medicinal wines that were popular for 2,000 or so years in early Egypt. News of the tasty mixture must have spread to Israel. “This wasn’t moonshine that someone was brewing in their basement, eyeballing the measurements,” Koh said. “This wine’s recipe was strictly followed in each and every jar.” The researchers hope to recreate the wine, so stay tuned for that, and... Two mysterious doors were found leading out of the wine cellar. They appear to lead to other rooms, but containing what? Maybe more wine? Another dig planned for 2015 should solve the mystery. (Image: Eric H. Cline, George Washington University) | Summary: Archeologists have found what they think is the oldest wine cellar on record-a 3,700-year-old collection of jars in the ruins of ancient Canaanite place in modern Israel, reports Bloomberg. They found about 40 ceramic jars, broken and empty but with the unmistakable residue of wine. Chemical analysis revealed that they were of both the red and white variety, and the New York Times ticks off ingredients including honey, mint, cinnamon bark, and juniper berries. The jars would have held enough for about 3,000 bottles. "It's a wine cellar that, to our knowledge, is largely unmatched in its age and size," says a lead researcher from George Washington University. It was discovered in the long-gone city of Tel Kabri, which was destroyed in some calamitous event-perhaps an earthquake-about 3,600 years ago. "The wine cellar was located near a hall where banquets took place, a place where the Kabri elite and possibly foreign guests consumed goat meat and wine," says an Israeli researcher on the project. Another dig is scheduled for 2015 to explore two doors leading out of the wine cellar, reports Discovery, meaning it's possible the wine cellar might be even bigger than thought. | 1,624 | 291 | multi_news | en |
Summarize: Just in case you weren't jealous enough of the French already, what with their effortless style, lovely accents and collective will to calorie control, they have now just banned bosses from bothering them once the working day is done. Well, sort of. Après noticing that the ability of bosses to invade their employees' home lives via smartphone at any heure of the day or night was enabling real work hours to extend further and further beyond the 35-hour week the country famously introduced in 1999, workers' unions have been fighting back. Now employers' federations and unions have signed a new, legally binding labour agreement that will require employers to make sure staff "disconnect" outside of working hours. Under the deal, which affects around 250,000 employees in the technology and consultancy sectors (including the French arms of Google, Facebook, Deloitte and PwC), employees will also have to resist the temptation to look at work-related material on their computers or smartphones – or any other kind of malevolent intrusion into the time they have been nationally mandated to spend on whatever the French call la dolce vita. And companies must ensure that their employees come under no pressure to do so. Thus the spirit of the law – and of France – as well as the letter shall be observed. That's right. While we poor, pallid, cowering Brits scurry about, increasingly cowed by the threat of recession-based redundancy and government measures that privilege bosses' and shareholder comfort over workers' rights, the continentals are clocking off. While we're staring down the barrel of another late one/extra shift/all-nighter, across the Channel they're sipping sancerre and contemplating at least the second half of a cinq à sept before going home to enjoy the rest of that lovely "work/133-hours-per-week-of-life" balance. C'est all right pour some, quoi? • This article was amended on 11 April 2014. An earlier version stated that the labour deal would affect "a million employees" and require staff "to switch off their phones after 6pm". The deal obliges staff to "disconnect" from work calls and emails after working hours to ensure they receive the full minimum rest periods already mandated in French employment regulations but there is no particular time at which they are required to do so. While the deal was signed by unions representing 1 million employees, it will affect only 250,000 workers directly. The guidelines state that ministry staff should not be penalised for switching off their mobiles or failing to pick up messages out of hours. The move follows similar restrictions on out-of-hours email imposed by German firms including Volkswagen, BMW and Puma. VW stops forwarding emails to staff from its company servers half an hour after the end of the working day, while other firms have declared that workers are not expected to check email at weekends or in their free time. The labour ministry's rules only allow contact if the task cannot be postponed until the next working day. Managers should apply a principle of "minimum intervention" into workers' free time and keep the number of people whose spare time is disrupted as low as possible. The code is part of a broader agreement covering remote working. Ursula von der Leyen, the labour minister, told the Sueddeutsche Zeitung the rules had been drawn up to protect workers' mental health. The minister said that it was important for remote workers to know: "When they have to be available, and when they don't. They now have this clarity in black and white." "It's in the interests of employers that workers can reliably switch off from their jobs, otherwise, in the long run, they burn out," she said. The minister called on companies to set clear rules over the out-of-hours availability of their workers earlier this year, warning that: "technology should not be allowed to control us and dominate our lives. We should control technology." The culture of routinely checking emails in spare time came under the spotlight in July, when the chief executive of Switzerland's biggest telecoms group was found dead at his flat in a suspected suicide. In an interview in May, Carsten Schloter, boss of Swisscom, criticised the need to be permanently engaged. "The most dangerous thing that can happen is that you drop into a mode of permanent activity," he said. "When you permanently check your smartphone to see if there are any new emails. It leads to you not finding any rest whatsoever." French workers already have a 35-hour work week, five vacation weeks a year, and now, some aren’t allowed to be contacted by their employer after 6:00pm. A new deal has been agreed upon between tech industry workers and unions in France that no longer requires employees to answer work-related emails after 6 p.m., according to reports. The deal reportedly includes one million workers in digital and consultancy sectors in the country. Tech giants Google (GOOG) and Facebook (FB) have operations in France. This deal means workers won’t be under any pressure to respond to their higher-ups post-clocking out, and they can’t be reprimanded in the highly-unionized country. Pam Villarreal, U.S. labor expert at the National Center for Policy Analysis, calls the deals “absurd.” “Within the tech industry and digital consultancy sectors, there’s always something going wrong off the clock—when a computer goes down, it doesn’t go down between 8 a.m. and 5 p.m.” she says. “Even though workers overwhelmingly support this, it will be interesting to see how it pans out in terms of productivity—knowing people who work in the tech industry, it’s one of the most likely where something goes wrong after hours.” She adds the deal could bring the unintended consequence of higher labor costs in the country’s industry. “They may have to shift workers to after 5 p.m. to deal with these issues, so it may drive up the cost of labor.” French tech workers aren’t the only ones getting a reprieve from the stress of a never-ending workday, as a Swedish city is experimenting with a six-hour workday, in an effort to improve productivity and happiness among workers. France also famously shortened its workweek to 35 hours instead of the standard 40, in 2000. “They are likely trying to reduce the unemployment rate, which is at 8%, and almost 24% for those under 25,” Villarreal says of Sweden. “But we don’t know if the experiment will work—people will be trying to cram 8 hours of work into a 6-hour day.” Villarreal says Swedish companies may also turn to outsourcing if labor costs climb, explain that France’s labor costs increased as a result of its shortened work week. Neither move is likely to hit the U.S. anytime soon, Villarreal says, especially the no contact after work agreement. “Think of big tech corporations—they wouldn’t agree to not contacting their employees after 6,” she says. “Google and Facebook are [probably] not so welcoming of this change in France. If it happened here, companies would probably try to outsource to India or China, countries without such strict labor regulations.” | Summary: France already has a 35-hour work week, and a new rule is designed to make sure that it doesn't start shading toward 40 hours because of work-related email. The Guardian reports that the rule forbids workers from checking their phones or computers for work stuff after 6pm, and it forbids employers from pressuring them to do so. The move apparently doesn't affect all workers in France, but it does cover about 1 million workers in the tech industry-including French employees of Google and Facebook. At Fox Business, a US labor expert finds it hard to believe the IT industry can manage such a draconian shut-off time. "There's always something going wrong off the clock-when a computer goes down, it doesn't go down between 8am and 5pm." A post at Fast Company agrees that it might be tough to enforce, but adds that "it's a nice perk to have in any case." Elsewhere, Germany already has a similar rule in place, while Sweden is experimenting with a 30-hour work week. | 1,606 | 235 | multi_news | en |
Summarize: Alligator attacks and kills 28-year-old man ORANGE- by Haley Bull Investigators, a witness and friends are revealing the harrowing accounts of the first recorded fatal alligator attack in Texas and more about the victim. A justice of the peace tells KFDM News Tommie Woodward, 28, is from St. Louis, but moved to Orange with his twin brother. He's worked in the shipyards the past few years. "One minute he's there and then the next minute he's gone," Michelle Wright said. What started as a night of fun at Burkhart's Marina in Orange ended with the fatal attack early Friday morning. "I asked him please do not go swimming, there's a bigger alligator out here, just please stay out of the water," Wright, an employee at the restaurant, said. Workers report they spotted a ten-foot gator in the waters of Adams Bayou this week, prompting them to post a warning sign reading "No Swimming Alligators." Investigators said Woodward didn't pay attention to the posted warnings and pleas from Wright. "He said blank the alligators and thereupon jumped into the water," Orange County Justice of the Peace Rodney Price said. Price said the 28-year old was almost immediately attacked and another young girl jumped in after him. "Next thing I know this girl is screaming an alligators got him, an alligator's got him and I grab a flashlight trying to zoom it over the water, trying to find him," Wright said. "The next thing I know, I don't even know how long it was, I saw his body floating face down and then he's up there for a couple seconds and then he gets dragged back down and pulled off." Wright said he disappeared in the water. "I kept trying to figure out and calling his name and calling his name and he just, he was dead. And it's heartbreaking," Wright said. Two hours later, searchers found Woodward's body about 200 yards away. "I just want to know why, why, you knew better than to go swimming," friend Angela Hoffpauir said. It's a warning investigators want others to heed. "Whether it's a speed limit sign on a freeway, a fireworks message, whatever, heed the warning sign, follow that because a failure to heed that can result in a terrible tragedy for your family," Judge Price said. Now family and friends are left remembering a life taken in an instant. "If you're upset, you're hurting, he could make you smile, he could make you laugh, he was just a funny person," Wright said. Judge Price has ordered an autopsy. Alligator attacks and kills 28-year-old man Tommie Woodward was known to jump off the marina near his house every now and again to dip in the bayou flowing through Orange, a small town on the outskirts of Beaumont. But recently the marina's owner spotted an alligator he estimated to be about 12 feet long. This is gator country, after all, home to about half a million alligators in the tri-county area of Orange, Jefferson and Chambers alone. Allen Burkart, the 82-year-old marina owner, put up a large sign: "No Swimming: Alligator." But that didn't deter Woodward, who showed up early Friday with a young woman, telling the staff closing the marina's burger joint that he was jumping in. They begged him not to, but the 28-year-old did anyway and his friend followed. Suddenly, witnesses told police, a large alligator emerged from under the dock. "He went under one time, then he hollered at her to get out of the water," Burkart said. "Then he pulled him down the second time and that was it." Sheriff's deputies and park rangers found Woodward dead in the water more than two hours later at about 4:30 a.m. near the marina in the 1800 block of Mississippi Street. Orange Police Capt. Robert Enmon said he suffered major trauma to his left arm and puncture wounds to his upper left chest. The alligator attack, the second one in the region this week, is believed to be the first fatal encounter in Texas in at least two centuries. Nationwide, the last time anyone was killed by an alligator was when a suspected car burglar jumped into a pond in western Miami-Dade County in 2007 as he was running from police. Feeding alligators In all, there have been 22 fatal alligator attacks in Florida since 1948. With 1.3 million alligators, it has the largest population in the nation. In Texas, the last fatal attack that went documented occurred in 1836 when an alligator killed a man swimming across the mouth of the Trinity. "It's extremely rare," said Gary Saurage, a prominent alligator hunter and co-owner of Gator Country Adventure Park in Beaumont. There are about 8 million alligators nationwide, he said, and in southeast Texas, "anytime you're swimming in a public waterway you can bet you're swimming alongside alligators." To him, the fatal incident seems suspicious. He wonders if the alligator was being fed from the marina, noting how it appeared from the dock and instantly attacked, as if expecting food. Texas Parks and Wildlife officials caution that feeding alligators, a Class C misdemeanor punishable by up to a $500 fine, makes them lose their natural fear of humans, and they become not only accustomed to the act but more aggressive. Comments on social media suggest at least some alligators were possibly fed there, with Lori Tatom, for instance, noting on a KFDM news story of the attack on Facebook: "We go here alot and people are always feeding the gator. His nickname is marshmallow and he's ALWAYS there." Because it's so rare, Saurage said the incident should be investigated. "We only had one fatal attack (nationally) in the last eight years," he said. "If (the alligator) has been hand-fed and you throw something next to him, he's going to grab it. I will bet you my reputation that's what happened here." Burkart, the marina's owner, said the large alligator arrived only recently, though two smaller ones have called this corner of the bayou home for much longer. "I'm sure people threw them a marshmallow or a Cheeto," he said. "The little ones they were feeding. But the big one wasn't coming in close." The two recent attacks come after a spike of reported reptile sightings following the area's historic rainfall in May. On Sunday authorities said an alligator bit 13-year-old Kaleb Hurley on the arm and dragged him underwater near the bank of Mac Pond, just off Lake Charlotte Road in Chambers County. Kaleb's father, 42-year-old James Hurley, ran into the water to help his son, but though the alligator bit him on the leg, he kicked it until it let him go. The two were flown by Life Flight helicopter ambulance to Memorial Hermann Hospital, where Kaleb remains, though his family expects him to be released next week. "This has been such a traumatic experience, to say the least, and we hope no one else out there ever has to go through what we have been through these past several days," the family said in a statement. "Please be sure to research the areas you are visiting thoroughly, especially if they have alligators or other wildlife." Experts say the recent rainfall filled the estuaries, displacing many alligators into places where they're more likely to encounter humans. "Alligators aren't long-distance athletes. They can't stay in fast-moving water long," said Chris Stephens, a nuisance alligator control hunter in the Harris County region who goes by "Gator Chris." "When the rivers are swollen they move to still water." An aggressive season The reptiles are also especially vigilant right now in protecting their territory because their mating, breeding and nesting season runs from May through September. Male alligators are more likely to be aggressive to attract females, whereas the fairer sex is protecting their nesting sites, Stephens said. The state's unusually mild winters over the past five years has also meant more alligators survived the cold season, swelling their ranks. "Alligators like to hunt in the dark, and they respond to splashing in the water," Stephens said. "That's usually a dinner bell to them, so they go investigate." A memorial page for Woodward on Facebook quickly drew posts from friends of the slender, bearded redhead. One said he tried to project a tough guy exterior, but was kind and gentle and years ago had enjoyed singing in their high school choir. Meanwhile Burkart, who was born and raised in Orange, said he's still reeling from the tragedy at his marina, which he has owned for more than half his life. Destroyed by Hurricane Ike in 2008, he refashioned it into a family-friendly place, complete with a shuffle board table and games. "I've been here all my life and this is the first time something like this has happened," he said. "I'm still in shock." | Summary: The sign said: "No Swimming: Alligator." The waitress said: "Please do not go swimming, there's a bigger alligator out here, just please stay out of the water." The swimmer said: "F--- the alligators." And that's how Texas recorded its first alligator death in about two centuries yesterday, reports the Houston Chronicle and KFDM. It happened in Orange County near Louisiana when 28-year-old Tommie Woodward ignored all the warnings and jumped into the water at Burkart's Marina. A gator emerged from under the dock and got him almost immediately, the first such death in Texas since 1836, reports Lomi Kriel at the Chronicle. "He went under one time, then he hollered at her to get out of the water," recalls owner Allen Burkart, referring to a friend of Woodward's who also jumped in. "Then he pulled him down the second time and that was it." Burkart himself had put up the sign recently after spotting the 12-foot gator in the water. Woodward disregarded the sign and the pleas from the staff at a marina burger shop. "He said blank the alligators and thereupon jumped into the water," the Orange County Justice of the Peace tells KFDM. | 2,108 | 292 | multi_news | en |
Summarize: By. Reuters. The FBI is probing Herbalife Ltd, the nutrition and weight loss company that hedge fund manager William Ackman has called a pyramid scheme, sources familiar with the investigation said on Friday.The news, first reported by the Financial Times, sparked a sharp sell-off that sent the stock price down nearly 14 percent. One source briefed on the matter said that the investigation has been going on for some time, but declined to give further details. A spokesman for the Federal Bureau of Investigation in Boston declined to confirm or deny the investigation. So far no charges have been filed in the matter. Former Herbalife distributors reached by Reuters said they had been contacted by agents who were interested in finding out more about the multilevel marketing company's business practices, including how it recruits new members into its distribution scheme. FBI agents are also reviewing Herbalife documents obtained from former distributors, two other sources familiar with the matter said. Scam? A Herbalife logo is shown on a poster at a clinic in the Mission District in San Francisco, California on April 29, 2013. Herbalife said on Friday that the company has not received any inquiries from the FBI or the Justice Department about any investigation. But it would not be unusual for law enforcement to investigate a company without telling executives about it. The company has steadfastly denied running a pyramid scheme, where distributors earn more money for recruiting new members into the scheme than they do by selling products to consumers. News of the FBI probe comes roughly a month after Herbalife confirmed that the Federal Trade Commission had issued a civil investigative demand to the company to look more closely at how it works. In January, U.S. Senator Edward Markey called on the FTC and the Securities and Exchange Commission to investigate the company, in part because of allegations from civil rights groups that Herbalife unfairly targets minorities. Herbalife denies this. The company, which reported sales of $4.8 billion last year, employs a vast network of independent distributors who sell its powders and shakes in more than 80 countries, including China, the company's fastest growing market. For months Herbalife has been a battleground for heavy-hitting investors. Ackman, who runs $13 billion hedge fund Pershing Square Capital Management from New York, first called the company a fraud in December 2012 when he unveiled a $1 billion short position against the company. Since then, billionaire investors Carl Icahn, George Soros, Daniel Loeb and William Stiritz have taken the other side of the bet, helping push the stock price up 138 percent last year. Their involvement has comforted other investors that Herbalife will eventually get a clean bill of health, one investor said. Pyramid scheme? Former Herbalife distributors have reportedly been contacted by FBI agents to better understand the company's business practices. But Ackman is sticking by his forecast that the company's share price will eventually fall to zero after regulators start looking at the company more closely. His firm declined to comment on Friday. Pershing Square has published dossiers about Herbalife's top distributors, charging they lure in new recruits with videos featuring big houses and expensive cars even though company statistics show that the majority of distributors never earn any income from Herbalife. Michael Araujo, a Massachusetts resident who tried to set up his own Herbalife distribution business for a year after having been laid off by Bank of America, said his family lost roughly $85,000 on the scheme. He said he spent about $4,000 a month buying business leads and purchased $7,000 worth of Herbalife products. "They target people who are desperate," Araujo said in a telephone interview, about the company's sales tactics. Meanwhile Tish Rochin, a distributor in Texas who went from driving a truck to saying she became a millionaire thanks to Herbalife, said it is'simple but hard work' to become wealthy by selling the company's products. This year Herbalife's stock price has tumbled 34.6 percent, kicked lower first by news that Senator Markey was involving himself. Last month news that the FTC opened an investigation hastened the fall. On Friday, the company's stock price closed at $51.48 | Summary: FBI is reportedly probing Herbalife Ltd, a nutrition and weight loss company but so far no charges have been filed. Former company distributors have said they've been contacted by agents who wanted to know more about company's practices. FTC issued a civil investigative demand to see how Herbalife operates about one month ago. Civil rights groups have alleged Herbalife unfairly targets minorities. Herbalife made $4.8 billion in sales last year and uses wide network of independent distributors to sell products. | 928 | 109 | cnn_dailymail | en |
Summarize: NASA scientists have proposed a bold plan that could give Mars its atmosphere back and make the Red Planet habitable for future generations of human colonists. By launching a giant magnetic shield into space to protect Mars from solar winds, the space agency says we could restore the Red Planet's atmosphere, and terraform the Martian environment so that liquid water flows on the surface once again. Mars may seem like a cold, arid wasteland these days, but the Red Planet is thought to have once had a thick atmosphere that could have maintained deep oceans filled with liquid water, and a warmer, potentially habitable climate. Scientists think Mars lost all of this when its protective magnetic field collapsed billions of years ago, and solar wind – high-energy particles projected from the Sun – has been stripping the Red Planet's atmosphere away ever since. Now, new simulations by NASA suggest there could be a way to naturally give Mars its thick atmosphere back – and it doesn't require nuking the Red Planet into submission, as Elon Musk once proposed. Instead, the space agency thinks a powerful-enough magnetic shield launched into space could serve as a replacement for Mars's own lost magnetosphere, giving the planet a chance to naturally restore its own atmosphere. In new findings presented at the Planetary Science Vision 2050 Workshop last week, NASA's Planetary Science Division director, Jim Green, said launching an "artificial magnetosphere" into space between Mars and the Sun could hypothetically shield the Red Planet in the extended magnetotail that trails behind the protective field. "This situation then eliminates many of the solar wind erosion processes that occur with the planet's ionosphere and upper atmosphere allowing the Martian atmosphere to grow in pressure and temperature over time," the researchers explain in an accompanying paper. While the team acknowledges that the concept might sound "fanciful", they point to existing miniature magnetosphere research being conducted to protect astronauts and spacecraft from cosmic radiation, and think that the same technology on a larger scale could be used to shield Mars. "It may be feasible that we can get up to these higher field strengths that are necessary to provide that shielding," Green said in his presentation. "We need to be able then to also modify that direction of the magnetic field so that it always pushes the solar wind away." In the team's simulations, if the solar wind were counteracted by the magnetic shield, Mars's atmospheric losses would stop, and the atmosphere would regain as much as half the atmospheric pressure of Earth in a matter of years. As the atmosphere becomes thicker, the team estimates Mars's climate would become around 4 degrees Celsius (7.2 degrees Fahrenheit) warmer, which would be enough to melt carbon dioxide ice over the Red Planet's northern polar cap. If this happened, the carbon in the atmosphere would help to trap heat like it does on Earth, triggering a greenhouse effect that could melt Mars's water ice, giving the Red Planet back its liquid water in the form of flowing rivers and oceans. If all of this were to occur as the team anticipates – and admittedly, that's a pretty fantastical if – it's possible that, within the space of a couple of generations, Mars could regain some of its lost Earth-like habitability. "This is not terraforming as you may think of it where we actually artificially change the climate, but we let nature do it, and we do that based on the physics we know today," Green said. The team acknowledges that the plan is largely hypothetical at this point, but it's a pretty amazing vision for what might be possible in the years ahead. The researchers intend to keep studying the possibilities to get a more accurate estimate of how long the climate-altering effects would take. If the concept does prove workable, there's no telling just how much it would alter the prospects of colonising Mars in the future. "Much like Earth, an enhanced atmosphere would: allow larger landed mass of equipment to the surface, shield against most cosmic and solar particle radiation, extend the ability for oxygen extraction, and provide 'open air' green-houses to exist for plant production, just to name a few," the researchers explain. "If this can be achieved in a lifetime, the colonisation of Mars would not be far away." The findings were presented at the Planetary Science Vision 2050 Workshop. Artist's conception of a terraformed Mars. Credit: Ittiz/Wikimedia Commons NASA proposes a magnetic shield to protect Mars' atmosphere This week, NASA's Planetary Science Division (PSD) hosted a community workshop at their headquarters in Washington, DC. Known as the "Planetary Science Vision 2050 Workshop", this event ran from February 27th to March 1st, and saw scientists and researchers from all over the world descend on the capitol to attend panel discussions, presentations, and talks about the future of space exploration. One of the more intriguing presentations took place on Wednesday, March 1st, where the exploration of Mars by human astronauts was discussed. In the course of the talk, which was titled "A Future Mars Environment for Science and Exploration", Director Jim Green discussed how deploying a magnetic shield could enhance Mars' atmosphere and facilitate crewed missions there in the future. The current scientific consensus is that, like Earth, Mars once had a magnetic field that protected its atmosphere. Roughly 4.2 billion years ago, this planet's magnetic field suddenly disappeared, which caused Mars' atmosphere to slowly be lost to space. Over the course of the next 500 million years, Mars went from being a warmer, wetter environment to the cold, uninhabitable place we know today. Artist’s rendering of a solar storm hitting Mars and stripping ions from the planet’s upper atmosphere. Credits: NASA/GSFC This theory has been confirmed in recent years by orbiters like the ESA's Mars Express and NASA's Mars Atmosphere and Volatile EvolutioN Mission (MAVEN), which have been studying the Martian atmosphere since 2004 and 2014, respectively. In addition to determining that solar wind was responsible for depleting Mars' atmosphere, these probes have also been measuring the rate at which it is still being lost today. Without this atmosphere, Mars will continue to be a cold, dry place where life cannot flourish. In addition to that, future crewed mission – which NASA hopes to mount by the 2030s – will also have to deal with some severe hazards. Foremost among these will be exposure to radiation and the danger of asphyxiation, which will pose an even greater danger to colonists (should any attempts at colonization be made). In answer to this challenge, Dr. Jim Green – the Director of NASA's Planetary Science Division – and a panel of researchers presented an ambitious idea. In essence, they suggested that by positioning a magnetic dipole shield at the Mars L1 Lagrange Point, an artificial magnetosphere could be formed that would encompass the entire planet, thus shielding it from solar wind and radiation. Naturally, Green and his colleagues acknowledged that the idea might sounds a bit "fanciful". However, they were quick to emphasize how new research into miniature magnetospheres (for the sake of protecting crews and spacecraft) supports this concept: The proposed method for creating an artificial magnetic dipole at Mars’ L1 Lagrange Point. Credit: NASA/J.Green "This new research is coming about due to the application of full plasma physics codes and laboratory experiments. In the future it is quite possible that an inflatable structure(s) can generate a magnetic dipole field at a level of perhaps 1 or 2 Tesla (or 10,000 to 20,000 Gauss) as an active shield against the solar wind." In addition, the positioning of this magnetic shield would ensure that the two regions where most of Mars' atmosphere is lost would be shielded. In the course of the presentation, Green and the panel indicated that these the major escape channels are located, "over the northern polar cap involving higher energy ionospheric material, and 2) in the equatorial zone involving a seasonal low energy component with as much as 0.1 kg/s escape of oxygen ions." To test this idea, the research team – which included scientists from Ames Research Center, the Goddard Space Flight Center, the University of Colorado, Princeton University, and the Rutherford Appleton Laboratory – conducted a series of simulations using their proposed artificial magnetosphere. These were run at the Coordinated Community Modeling Center (CCMC), which specializes in space weather research, to see what the net effect would be. What they found was that a dipole field positioned at Mars L1 Lagrange Point would be able to counteract solar wind, such that Mars' atmosphere would achieve a new balance. At present, atmospheric loss on Mars is balanced to some degree by volcanic outpassing from Mars interior and crust. This contributes to a surface atmosphere that is about 6 mbar in air pressure (less than 1% that at sea level on Earth). At one time, Mars had a magnetic field similar to Earth, which prevented its atmosphere from being stripped away. Credit: NASA As a result, Mars atmosphere would naturally thicken over time, which lead to many new possibilities for human exploration and colonization. According to Green and his colleagues, these would include an average increase of about 4 °C (~7 °F), which would be enough to melt the carbon dioxide ice in the northern polar ice cap. This would trigger a greenhouse effect, warming the atmosphere further and causing the water ice in the polar caps to melt. By their calculations, Green and his colleagues estimated that this could lead to 1/7th of Mars' oceans – the ones that covered it billions of years ago – to be restored. If this is beginning to sound a bit like a lecture on how to terraform Mars, it is probably because these same ideas have been raised by people who advocating that very thing. But in the meantime, these changes would facilitate human exploration between now and mid-century. "A greatly enhanced Martian atmosphere, in both pressure and temperature, that would be enough to allow significant surface liquid water would also have a number of benefits for science and human exploration in the 2040s and beyond," said Green. "Much like Earth, an enhanced atmosphere would: allow larger landed mass of equipment to the surface, shield against most cosmic and solar particle radiation, extend the ability for oxygen extraction, and provide "open air" greenhouses to exist for plant production, just to name a few." These conditions, said Green and his colleagues, would also allow for human explorers to study the planet in much greater detail. It would also help them to determine the habitability of the planet, since many of the signs that pointed towards it being habitable in the past (i.e. liquid water) would slowly seep back into the landscape. And if this could be achieved within the space of few decades, it would certainly help pave the way for colonization. In the meantime, Green and his colleagues plan to review the results of these simulations so they can produce a more accurate assessment of how long these projected changes would take. It also might not hurt to conduct some cost-assessments of this magnetic shield. While it might seem like something out of science fiction, it doesn't hurt to crunch the numbers! Explore further: NASA awards launch services contract for Mars 2020 rover mission More information: www.hou.usra.edu/meetings/V2050/pdf/8250.pdf The concept isn't as far-fetched as it sounds. There's already research into inflatable structures that would create the mini-magnetosphere you'd need for this to work. The biggest challenge, as with many things in space, is time. While a shield would have a relatively rapid effect on radiation, it's not certain how long it would take to thicken the atmosphere and increase temperatures. A magnetic shield would amount to a kind of terraforming, after all, and even a relatively quick change could take decades. NASA and SpaceX would still have to visit a hostile planet in the years ahead. Still, the very fact that this is even on the table is noteworthy. It suggests that it would be realistic to preserve and even improve Mars' remaining atmosphere without the staggering effort that would be needed to directly alter the planet. | Summary: Mars isn't particularly habitable to humans at the moment, but NASA's latest brainstorm could one day bring back the planet's beaches-or at least some of its oceans, Engadget reports. At a workshop in DC last week, scientists from the agency's Planetary Science Division presented the possibility of erecting a magnetic shield around the Red Planet to replace the one that vanished more than 4 billion years ago and boost what's left of Mars' atmosphere, per Phys.org. The result of such a shield, according to the researchers, would be protection from solar radiation and winds, which have worked over the eons to help strip the planet of its waters and warmth. The shield could, therefore, help "terraform" Mars to more closely resemble Earth. In a plan some say is "so crazy it just might work," the shield (maybe an inflatable one) would be placed at the L1 Lagrange Point. It would hopefully thicken the atmosphere to the point where carbon dioxide ice at the planet's north pole would thaw, spur greenhouse-gas warming, and melt regular ice, which would restore up to one-seventh of Mars' oceans, according to NASA estimates. Per the scientists' report on what they admit is a "fanciful" topic, this "enhanced" atmosphere would be more conducive to human exploration, offering more possible oxygen extraction, more plant growth in "open-air" greenhouses, and the ability of larger structures to land on the planet's surface. | 2,712 | 332 | multi_news | en |
Summarize: Friends and family are grieving the loss of Brendan Hickey after he drowned at Sydney's Darling Harbour late on Friday night. Police were told that the Irishman, who lived in Randwick, was sitting with three of his friends on the edge of a wooden pier to watch Sydney's Vivid light festival when he disappeared into the water just before 11pm. A female friend and another man dove into the water fully clothed in a desperate bid to save him, but were unsuccessful in locating the 34-year-old. Tributes have poured in for Irishman Brendan Hickey (right), pictured here with Julia Szymanska. The pair emerged from the water unharmed and police recovered Brendan's body about three metres out from the boardwalk at Cockle Bay Wharf at 1.30am on Saturday. Tributes have poured in for the Irish 'gentleman'. After his brother Shane Hickey posted a photo of the pair of them smiling in green t-shirts on his Facebook page one friend, Suzanne Colgan, wrote:. 'My thoughts are with you and your family... Bren was one of the greatest guys u knew, I'm devastated.' Scroll down for video. Distraught friends are interviewed by police after the Randwick resident fell into the water at Sydney's Darling Harbour on Friday night. An extensive search was launched by emergency services who recovered the man's body at 1.30am on Saturday. Marianne Hickey wrote: 'So sorry Shane. I have no words. I'm thinking of u cuz....il c u soon xxxxxx' 'Thinking of you and all the family! He was an absolute gentleman!,' Adrian McGeown said. A woman who appears in numerous photos with Brendan on his Facebook page, Julia Szymanska, 22, has also posted a tribute photo of her and him together, on Saturday. Friend Fiona Syombua, commented: 'I cannot believe this sad news. Brendan Hickey very happy, ever smiling. gone too soon. I will always live to remember you. my heart is in deep mourn right now.' Mr Hickey posted this photo of him with Julia Szymanska in April from Nelson Bay Beach, NSW. Shocked: Family and friends have expressed their grief on Brendan's brother Shane Hickey's Facebook page. Police confirmed Brendan accidentally fell into the water. A family member told Nine News that he couldn't swim. However, acting Superintendent Joe McNulty, Commander of the Marine Area Command said reports that Mr Hickey couldn't swim have not been confirmed. A helicopter flew above Darling Harbour to help search for the missing man. 'His friends have been very emotionally affected by the incident which hasn’t helped us with the investigation to date,' he told reporters. 'Witnesses said they heard a splash and they saw the 34-year-old man in the water.' Shocked onlooker Mark Jacoub watched the frantic search unfold. 'We saw the girl jump off the police boat first then another guy jumped in after her,' he told the MailOnline. Brothers: Shane Hickey posted this photo of Brendan (left) on Saturday as tributes poured in. Tragic: Brendan (left) is seen here with friends at the Ivy nightclub in Sydney's CBD in February. Accident: Brendan's brother believes his death was an accident as the 34-year-old man couldn't swim. 'They were in the water for less than 40 seconds.' Onlooker Stephan Odisho told the Telegraph: 'At first I thought someone had lost their wallet, and then it got really urgent with people jumping in.' Witnesses said security men then came over to tell the two friends to get out of the water without realising what had just happened. An extensive helicopter search was launched at 11pm while Police Rescue, Sydney Harbour Foreshore Authority staff, security from nearby Home Nightclub and police officers joined forces to help look for the man by shining their torches into the pitch black water. Water police desperately try to find the man in the pitch black water. Shocked onlookers watched the frantic search unfold. Vivid Sydney'shocked and saddened' by the drowning incident which took place on the launching night of the light festival. Police are also investigating whether or not alcohol was a factor. Vivid Sydney expressed their condolences via their Twitter account later on Saturday afternoon. 'We are shocked and saddened by the tragic incident last night and express our condolences to family and friends of the deceased man,' festival organisers wrote. Inquiries into the matter are continuing as CCTV footage is being reviewed. Police urged anyone who saw the incident to come forward. It is believed the man was sitting at the wooden pier watching Vivid Sydney, an annual light festival. Sorry we are not currently accepting comments on this article | Summary: Brendan Hickey, an Irishman who lived in Randwick, was watching the light show with his friends when he accidentally fell into Sydney's Darling Harbour just before 11pm on Friday. His female friend and another man jumped into the water and desperately tried to find the 34-year-old. An extensive helicopter search was launched and police recovered the man's body at 1.30am on Saturday. | 1,107 | 97 | cnn_dailymail | en |
Summarize: This is a continuation of application Ser. No. 08/128,629, filed Sep. 29, 1993, now abandoned. BACKGROUND OF THE INVENTION The present invention relates to the field of ophthalmology. More specifically, the invention relates to the field of cataract surgery, wherein the natural crystallin lens of the human eye is surgically removed and an artificial lens is implanted. Modern cataract surgery typically involves implantation of an artificial lens, referred to as an "intraocular lens" or "IOL", in the posterior chamber of the eye. The preferred site of implantation is within the capsule which surrounds the natural crystallin lens. When the natural crystallin lens is surgically removed, a portion of the anterior face of the lens capsule is also removed. This provides an opening which allows the artificial lens to be placed within the remaining portion of the lens capsule, which is also referred to as the "capsular bag". The capsular bag is considered to be the ideal location for implantation of an intraocular lens. Unfortunately, there is a significant problem associated with implantation of intraocular lenses in the capsular bag. The capsular bag is normally cleaned or "polished" by the ophthalmic surgeon to remove lens epithelial cells and other tissue remnants. This helps to ensure that deposits in the lens capsule do not impair the vision of the patient. However, it is generally not possible for the surgeon to remove all of the lens epithelial cells, particularly in the outer perimeter of the capsular bag. The remaining lens epithelial cells may eventually cause opacifications which impair the vision of the patient. Such impairment is referred to as "secondary cataract". The formation of a secondary cataract may require further medical treatment, such as use of a YAG laser to break up the opacifications, or further surgery. In the past, the use of antimetabolites, such as 5'-fluorouracil or mitomycin C, has been suggested as a means of preventing secondary cataract formation. Although antimetabolites can prevent the growth and proliferation of active lens epithelial cells, this approach has not proved to be effective. It is believed that the effectiveness of antimetabolites in preventing lens epithelial cell growth is severely limited by the fact that these agents only target cells that are actively dividing. Since the antimetabolites are normally applied to the lens capsule in a single dose at the time of surgery, it is not possible for these agents to prevent the subsequent proliferation of lens epithelial cells which are dormant (i.e., not actively dividing) at the time of surgery. A significant number of lens epithelial cells may therefore evade the action of the antimetabolite and proliferate at a later time. This lens epithelial cell proliferation ultimately contributes to the formation of lens opacifications or secondary cataracts. Thus, there is a need for an improved method of preventing or retarding the formation of secondary cataracts. The present invention is directed to satisfying this need. SUMMARY OF THE INVENTION The present invention provides an improved method of preventing or retarding secondary cataract formation. The method is based on the application of a composition which contains a combination of one or more lens epithelial cell growth stimulators and an antimetabolite to the lens capsule at the time of surgery. Various lens epithelial cell growth stimulators or combinations thereof may be utilized for this purpose, but the most preferred approach is to utilize a combination which includes transforming growth factor-beta ("TGF-β"). The lens epithelial cell stimulator component of the composition stimulates lens epithelial cells which are dormant at the time of surgery, so as to cause these cells to initiate DNA synthesis. This mitogenic activation of the lens epithelial cells enhances their susceptability to the action of the antimetabolite component of the composition. The above-described method results in a much greater suppression of lens epithelial cell growth, compared to that achieved with an antimetabolite alone. As a result, the method provides a significant improvement in the ability to prevent or retard the formation of secondary cataracts. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1, the sole figure of drawings, is a bar graph presentation of the data discussed in Example 1. DESCRIPTION OF PREFERRED EMBODIMENTS The lens epithelial cell stimulators which may be utilized in the present invention include all agents which will activate dormant lens epithelial cells by stimulating the initiation of DNA synthesis. Such agents are collectively referred to herein as "lens epithelial cell growth stimulators". The use of mixtures which include various isoforms of TGF-β, and modifications thereof, is preferred. There are five known isoforms of TGF-β. These forms have been designated as TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 and TGF-β 5, the first three being common to man. The physical properties of these growth factors, sources of same, and methods of purification are known. See, for example, U.S. Pat. No. 5,108,989 (Amento, et al; Genentech, Inc.) and the references cited therein at lines 21-45 of column 1. The entire contents of that patent relating to the various forms of TGF-β are hereby incorporated by reference in the present specification. As utilized herein, the term "TGF-β" encompasses one or more polypeptides having lens epithelial cell stimulating activity, such as mature and precursor forms of TGF-β 1, TGF-β 2, TGF-β 3, TGF-β 4 and TGF-β 5 ; hybrid TGF-βs; latent TGF-β complexes; TGF-β analogs (e.g., deletion variants and hybrids); and biologically active polypeptides based on transforming growth factor-beta sequences, such as those described in U.S. Pat. No. 5,061,786 (Burnier, et al.; Genentech, Inc.). The lens epithelial cell growth stimulators which may be utilized in the present invention also include transforming growth factor-alpha (TGF-∝), keratinocyte growth factor (KGF), epidermal growth factor (EGF), platelet-derived growth factors (PDGF-BB, -AA, or -AB), basic fibroblast growth factor (b-FGF), acid fibroblast growth factor (a-FGF), angiogenin, nerve growth factor (NGF), insulin-like growth factor I and II (IGF-I and IGF-II), and other proteins or polypeptides having mitogenic activity relative to lens epithelial cells. As used herein, the term "polypeptides" encompasses natural, synthetic and recombinant polypeptides, including polypeptides having deleted, replaced or altered amino acid sequences in comparison with the full-length natural polypeptide or biologically active fragments thereof. The lens epithelial cell growth stimulators utilized in the present invention are preferably human derived. As used herein, the term "human derived" encompasses agents recovered from human tissues and agents produced from human cell lines by means of recombinant DNA technology. The most preferred lens epithelial cell growth stimulator of the present invention is a mixture which includes TGF-β, in combination with EGF, b-FGF, TGF-∝ and PDGF-BB. The compositions utilized in the present invention contain one or more of the above-described lens epithelial cell stimulators in an amount sufficient to achieve mitogenic activation of dormant lens epithelial cells. The amount of lens epithelial cell stimulator required for this purpose will vary depending on the particular agent(s) utilized, but will generally be from about 0.01 to about 10,000 nanograms/milliliter ("ng/ml"). The compositions utilized in the present invention will also include one or more antimetabolites to suppress the proliferation of lens epithelial cells. Various antimetabolites may be utilized for this purpose. The antimetabolites which may be utilized can be generally characterized as being structural analogs to metabolically active molecules, such as purines, pyrimidines and folic acid. These compounds interfere with normal DNA/RNA synthesis, and thereby terminate cell growth. The net effects of this action are reduced mitotic activity and impaired proliferation of lens epithelial cells. Examples of antimetabolites which may be utilized in the present invention include mitomycin C, 5'-fluorouracil, arabinocytosine, taxol, actinomycin C and methotrexate. The use of mitomycin C as the antimetabolite component of the present invention is preferred, because it is relatively less toxic to corneal endothelial cells than other antimetabolites, such as 5'-fluorouracil. The compositions utilized in the present invention contain one or more of the above-described antimetabolites in an amount sufficient to suppress the growth of lens epithelial cells. The amount of antimetabolite required for this purpose will vary depending on the particular antimetabolite(s) selected, but will generally be from about 0.01 to about 500 micrograms/milliliter ("mcg/ml"). The above-described combinations of lens epithelial cell stimulators and antimetabolites can be included in various types of pharmaceutical vehicles suitable for intraocular use. The vehicles are preferably aqueous, and are formulated so as to be chemically and physically compatible with ophthalmic tissues. For example, viscoelastic formulations currently utilized in connection with intraocular surgical procedures, such as HEALON® (sodium hyaluronate) (Kabi Pharmaci Ophthalmics Inc.) or VISCOAT® (sodium chondrotin sulfate-sodium hyaluronate) Sterile Ophthalmic Viscoelastic (Alcon Laboratories, Inc.) (Alcon Surgical, Inc.) may be utilized as a vehicle for the above-described combinations of growth factors and antimetabolites. Such viscous formulations are coherent and tend to adhere to tissue. These properties help to ensure that the compositions will expose lens epithelial cells to the actions of the growth factor/antimetabolite combinations of the present invention. This is particularly true when the compositions are applied following removal of the natural crystallin lens, since at that point the capsular bag will be at least partially open and therefore prone to immediately losing any fluid which is applied to the interior of the bag by means of irrigation. The use of a viscous solution or semi-solid composition may therefore be preferable in some cases. In other cases, such as those where the growth factor/antimetabolite combination is injected into the capsular bag prior to removal of the natural crystallin lens, the viscosity of the composition will not be a primary concern, since leakage of the composition from the capsular bag will be a less significant problem. The use of an aqueous solution as the vehicle for the growth factor/antimetabolite combination may therefore be preferred in such cases. The aqueous solutions which might be utilized must be compatible with intraocular tissues, and should preferably help to maintain the integrity and function of intraocular tissues during the surgical procedure. The aqueous solutions which might be utilized for the above-described purposes include balanced saline solutions, such as BSS® Balanced Salt Solution and BSS Plus® Balanced Salt Solution Enriched with Bicarbonate, Dextrose and Glutathione, both of which are available from Alcon Surgical, Inc., and BION™ Tears Lubricant Eye Drops (Alcon Laboratories, Inc.), which is available from Alcon Laboratories, Inc., Fort Worth, Tex. As will be appreciated by those skilled in the art, the above-described compositions must be sterile and should not include any agents (e.g., antimicrobial preservatives) which will be toxic to sensitive intraocular tissues, particularly corneal endothelial cells. The above-described compositions can be formulated in accordance with techniques known to those skilled in the art. The following publications may be referred to for further details concerning the formulation of compositions containing polypeptides, such as TGF-β: U.S. Pat. No. 4,717,717 (Finkenaur; Ethicon, Inc.); U.S. Pat. No. 4,962,091 (Eppstein, et al.; Syntex (U.S.A.) Inc.); and WO 92/15614 (Takruri; Chiron Ophthalmics, Inc.); and references cited in the foregoing patent publications. The above-described compositions can be applied to the lens capsule by means of various techniques. For example, the compositions can be applied to the interior of the capsular bag by means of a syringe following removal of the crystallin lens, or can be injected into the lens capsule prior to removal of the crystallin lens by means of phacoemulsification or other methods. The only critical requirement with respect to how the compositions are applied is that the compositions be distributed throughout the lens capsule, and remain in contact with the dormant lens epithelial cells for a length of time sufficient to achieve mitogenic activation of those cells. The amount of time required to achieve this purpose will vary somewhat depending on circumstances such as the lens epithelial cell stimulators and antimetabolites utilized, and the method by which the lens epithelial cell stimulator/antimetabolite combination is applied to the capsular bag (i.e., injection prior to phacoemulsification or irrigation following cataract removal). However, the compositions will generally need to remain in contact with the interior of the capsular bag for approximately five to ten minutes or longer. The compositions may be removed by means of conventional irrigation and aspiration techniques. The following example is provided to illustrate the effect of the above-described lens epithelial cell stimulators and antimetabolites on lens epithelial cells. EXAMPLE 1 Studies were conducted to examine the effects of a particular antimetabolite, mitomycin C, on the mitogenic capability of cultured rabbit lens epithelial cells. A standard in vitro mitogenesis assay was utilized for these experiments. Normal rabbit lens epithelial cells (designation AG04676 from the Coriell Institute for Medical Research Cell Repository) between passage number six and thirteen were plated in a 96-well tissue culture plate. The cells were fed fresh MEM Eagle medium with Earle's salts supplemented with 10% normal rabbit serum, 2 mM 1-glutamine, and 0.05 mg/ml gentamicin every three to four days until the cells were approximately 80% confluent. The experiment was initiated three to five days after the last medium replacement to assure that the cells were at their maximum mitogenic potential. The test compounds (growth factor mixture and antimetabolite), formulated in serum-free culture medium, were exposed to cells in triplicate wells for ten minutes. The test substances were then aspirated off of the cells and replaced by serum-free culture medium. After an eighteen hour incubation, tritiated thymidine was exposed to the cells for six hours. The DNA was then precipitated with trichloroacetic acid; thymidine not incorporated into DNA was removed by washing, and the labeled DNA was solubilized with sodium dodecyl sulfate. The tritiated thymidine which was incorporated into the DNA of actively dividing cells was quantitated using a beta scintillation counter, and the mean and standard deviation were calculated for the triplicate sets. In these particular studies, a mixture of growth factors was used to maximally stimulate mitogenesis in these cells. The mixture consisted of 30 ng/ml of epidermal growth factor and 20 ng/ml each of platelet-derived growth factor (-BB homodimer), basic fibroblast growth factor, and transforming growth factor-alpha. As depicted in FIG. 1, the addition of 20 ng/ml of transforming growth factor-beta significantly increased the stimulatory capability of the growth factor mixture. Mitomycin C was added to the cells in the presence of the growth factor mixture at various concentrations. The results presented in FIG. 1 indicate that the cells exposed to the growth factor mixture were more active in synthesizing DNA, when compared to control (i.e., unstimulated) cells. In addition, TGF-β 1 by itself has little or no effect on cell growth; however, when added to the growth factor mixture, TGF-β 1 enhances the DNA synthesis stimulatory effect. The mitogenic mixture which included TGF-β 1 maximized the stimulation of lens epithelial cell DNA synthesis. This stimulatory effect was effectively inhibited by mitomycin C (MIT C) at concentrations above 2.5 ug/ml (inhibition=75.5%). It is important to note that the growth factor mixture and mitomycin C were only exposed to the cells for a ten minute period. | Summary: The intraocular use of combinations of lens epithelial cell growth stimulators (e.g., TGF-β) and antimetabolites (e.g., mitomycin C) is described. The combination is applied to the capsular bag to prevent or retard the formation of secondary cataracts following cataract surgery. The lens epithelial cell stimulators activate DNA synthesis in dormant lens epithelial cells, and thereby make those cells susceptible to the anti-metabolites. This enables the antimetabolites to suppress the proliferation of lens epithelial cells to a much greater extent, relative to the proliferation observed when the metabolites alone are utilized. The increased suppression of the growth of lens epithelial cells results in a significant improvement in the ability to prevent or retard the formation of opacities on the lens capsule (i.e., secondary cataracts). | 3,919 | 191 | big_patent | en |
Summarize: This Issachar was the most choleric Hebrew that had ever been seen in Israel since the Captivity in Babylon. "What!" said he, "thou bitch of a Galilean, was not the Inquisitor enough for thee? Must this rascal also share with me?" In saying this he drew a long poniard which he always carried about him; and not imagining that his adversary had any arms he threw himself upon Candide: but our honest Westphalian had received a handsome sword from the old woman along with the suit of clothes. He drew his rapier, despite his gentleness, and laid the Israelite stone dead upon the cushions at Cunegonde's feet. "Holy Virgin!" cried she, "what will become of us? A man killed in my apartment! If the officers of justice come, we are lost!" "Had not Pangloss been hanged," said Candide, "he would give us good counsel in this emergency, for he was a profound philosopher. Failing him let us consult the old woman." She was very prudent and commenced to give her opinion when suddenly another little door opened. It was an hour after midnight, it was the beginning of Sunday. This day belonged to my lord the Inquisitor. He entered, and saw the whipped Candide, sword in hand, a dead man upon the floor, Cunegonde aghast, and the old woman giving counsel. At this moment, the following is what passed in the soul of Candide, and how he reasoned: If this holy man call in assistance, he will surely have me burnt; and Cunegonde will perhaps be served in the same manner; he was the cause of my being cruelly whipped; he is my rival; and, as I have now begun to kill, I will kill away, for there is no time to hesitate. This reasoning was clear and instantaneous; so that without giving time to the Inquisitor to recover from his surprise, he pierced him through and through, and cast him beside the Jew. "Yet again!" said Cunegonde, "now there is no mercy for us, we are excommunicated, our last hour has come. How could you do it? you, naturally so gentle, to slay a Jew and a prelate in two minutes!" "My beautiful young lady," responded Candide, "when one is a lover, jealous and whipped by the Inquisition, one stops at nothing." The old woman then put in her word, saying: "There are three Andalusian horses in the stable with bridles and saddles, let the brave Candide get them ready; madame has money, jewels; let us therefore mount quickly on horseback, though I can sit only on one buttock; let us set out for Cadiz, it is the finest weather in the world, and there is great pleasure in travelling in the cool of the night." Immediately Candide saddled the three horses, and Cunegonde, the old woman and he, travelled thirty miles at a stretch. While they were journeying, the Holy Brotherhood entered the house; my lord the Inquisitor was interred in a handsome church, and Issachar's body was thrown upon a dunghill. Candide, Cunegonde, and the old woman, had now reached the little town of Avacena in the midst of the mountains of the Sierra Morena, and were speaking as follows in a public inn. | Summary: Issachar sees Candide with Cunegonde and attacks him with his sword. Candide, conveniently armed, attacks and kills Issachar. Candide and Cunegonde consult the Old Woman about what to do now that they have a dead body on their hands. In the middle of their deliberation, the Inquisitor comes into the room to visit with his sex slave. Candide thinks fast and kills the Inquisitor before he has time to recover from his surprise. That's two men dead. Cunegonde is horrified. The Old Woman suggests a quick escape by horseback. She's a little nervous about her own ability to ride, since she apparently has only one buttock. They escape on horseback and arrive at an inn in a small town called Avacena. | 764 | 175 | booksum | en |
Summarize: A compound built for notorious cult leader, polygamist and child abuser Warren Jeffs has been turned into a bed and breakfast. Fittingly for a property built for a man who once made the FBI’s list of 10 most wanted fugitives, the guesthouse in Hildale, Utah has been renamed Most Wanted Suites. Construction of the ranch began in 2010 while Jeffs was awaiting trial in Texas for sexual assault and aggravated sexual assault of a child in the hope he would cleared of his crimes and could live there with his wives and children. But the leader of Fundamentalist Church of Jesus Christ of Latter-Day Saints was convicted in 2011 and sentenced to life in prison plus 20 years, with the ranch subsequently seized by the government. Bed and breakfast: Fittingly for a property built for a man who once made the FBI's list of 10 most wanted fugitives, the guesthouse in Hildale, Utah (pictured) has been renamed Most Wanted Suites. Sick: A compound built for notorious cult leader and polygamist Warren Jeffs (pictured left and right) while he was awaiting trial in Texas for sexual assault and aggravated sexual assault of children. After Jeff’s conviction, the newly-built compound was seized by the U.S. government and later put up for auction. The winner of that auction was Willie Jessop – a former member of the church and a confidante of Jeffs, who later sued the group and won. Jessop purchased the ranch with a bid of $3.6million during an auction in April 2013, according to the Salt Lake Tribune. Now he says he is using the guesthouse as a way of helping the local community to come to terms with the past. ‘The initial intent of the [the compound] was to be something exclusive, and the bed and breakfast concept made it something inclusive,’ he told the newspaper. The cost of a stay at Most Wanted Suites is not listed on the company website, but each room comes with a private bathroom, and breakfast will either be served on site or at the Merry Wives Café down the road. Jessops says the best room – a large circular space once intended as a bedroom for Jeffs himself – will be off limits for some time as it is currently being used to house a family whose property recently burned down. Renewal: After Warren Jeff’s 2011 conviction for sexual assault and aggravated sexual assault of children, the newly-built compound was seized by the U.S. government and put up for auction. Women in the sect: The brides of polygamist cult leader Warren Jeffs. He was sentenced to life in prison for the sexual assault of underage girls. The presence of the controversial church is never far from the ranch however, as both Hildale and neighbouring Colorado City in Arizona have high rates of membership. Indeed the opening of the twin towns’ first bed and breakfast is part of a state-sponsored drive to boost a local economy hit by rising unemployment caused by rapidly dwindling membership of the church since Jeffs was jailed. Most Wanted Suites has also already been used for a local function – albeit a reunion for former pupils of a high school run by Jeffs’ church. Jeffs himself was born into the FLDS church, with his father Woodruff Steed the former leader. He was one of at least 60 children of Steed, who was survived by approximately 20 wives. The Utah-based church believes polygamy brings exaltation in heaven and arranged marriages sometimes involve underage girls. Upon Steed’s death Jeffs married all but two of his wives and instructed them to act as if his father was ‘still alive and in the next room.’ When Jeffs rose to lead the FLDS himself, he was given the title President and Prophet, Seer and Revelator’, and was also referred to a ‘President of the Priesthood. Innocent victims: Children living in Warren Jeffs' compound are taught that the self-proclaimed prophet convicted of child sex abuse is the U.S. president. Previous base: Much of the FLDS' activity took place on the Yearning for Zion ranch (pictured), which is located just outside the town of Eldorado in Texas. A 2008 raid on the stronghold revealed the polygamous community which included pregnant child brides in pastel prairie dresses with elaborate braided hairdos. As the sole individual allowed to conduct marriage ceremonies, Jeffs is believed to have been responsible for arranging hundreds of marriages between members. Running the towns as a form of overlord, Jeffs even expelled 20 men – including the mayor – from Colorado City, before ‘reassigning’ their wives. Much of the FLDS’ activity took place on the Yearning for Zion ranch, which is located just outside the town of Eldorado in Texas. It was home to at least 500 members of the controversial church until as recently as April 2014, when the state of Texas seized it on suspicion the property was being used ‘to commit of facilitate certain criminal conduct’. A 2008 raid on the stronghold revealed the polygamous community which included. pregnant child brides in pastel prairie dresses with elaborate braided. hairdos. Jeffs himself was placed on the FBI’s most wanted list in May 2006 after he took flight while facing charges relating to arranging illegal marriages between adult male followers and underage girls. He was arrested three months later in Nevada and hit with eight additional charges, including sexual conduct with minors and incest. Following years of court cases – including a 10-year conviction for rape as an accomplice which was subsequently overturned – Jeffs was finally found guilty of sexual assault and aggravated sexual assault of children and sentenced to life in prison plus 20 years | Summary: Ranch in Hildale, Utah was built while Jeffs awaited child sex abuse trial. Cult hoped he would one day live there with his many wives and children. But the notorious polygamist was found guilty and jailed for life in 2011. Now the compound has been transformed into a 14-bedroom guesthouse. New owner Willie Jessop is a former member of Jeffs' controversial church. He hopes to help local community recover from the cult leader's crimes. | 1,309 | 112 | cnn_dailymail | en |
Summarize: Oscar Pistorius, the Paralympic athlete accused of murdering his girlfriend, has broken his long public silence to describe her death as a "devastating accident". Pistorius issued a statement to mark the first anniversary of the Valentine's day tragedy in which he shot Reeva Steenkamp three times through a toilet door at his home in Pretoria, South Africa. He maintains that he believed her to be an intruder. Just after 1am local time on Friday, Pistorius tweeted for the first time in a year, linking to the statement on his website. "No words can adequately capture my feelings about the devastating accident that has caused such heartache for everyone who truly loved – and continues to love Reeva," it said. "The pain and sadness, especially for Reeva's parents, family and friends consumes me with sorrow. The loss of Reeva and the complete trauma of that day, I will carry with me for the rest of my life – Oscar." Pistorius, known as the Blade Runner because of his prosthetic legs, has kept a low profile since being released on bail last year, staying at the home of his uncle and resuming "low-key" training. He is due to stand trial for murder on 3 March in a case that has attracted huge worldwide attention. Steenkamp's parents, Barry and June, also issued a statement this week through their lawyers. "All we are looking for is closure and to know that our daughter did not suffer on that tragic Valentine's day," they said. "As the first anniversary of our beloved daughter's death approaches, we would like to thank all family, friends, the people of South Africa, and the world for their compassion, kind words and comfort shown to us, and for the many letters of condolences we have received." The family added that they intend to set up a foundation in honour of Steenkamp after the trial. "Reeva, who held such a passion for women's abuse issues and frequently spoke out against domestic violence, intended to one day open an establishment where abused women would be cared for." Steenkamp's schoolfriend Gwyn Guscott posted on Facebook on Thursday: "Tomorrow will be a tough one for most, but let's remember how much she loved love and fill our day with roses and remember her stunning smile and that little giggle she used to do when she had happy thoughts. To Reeves, I miss you every day." 14 February 2014 No words can adequately capture my feelings about the devastating accident that has caused such heartache for everyone who truly loved – and continues to love Reeva. The pain and sadness – especially for Reeva’s parents, family and friends consumes me with sorrow. The loss of Reeva and the complete trauma of that day, I will carry with me for the rest of my life. – Oscar Following the tragic event and the enormous global interest, the family of Oscar Pistorius has taken the decision to devote his official website to the latest news about developments as well as messages of support. The website will provide the opportunity for the media to make enquiries or requests but for understandable legal reasons it may not always be possible to respond or comment. The Pistorius family and Oscar’s management company have been inundated with messages of support and condolences for Oscar and for the family of Reeva Steenkamp from all over the world. Mr Arnold Pistorius, uncle of Oscar, said on behalf of the family: “We believe that this is an appropriate way to deal with the expressions of support we have received as well as keeping the media informed about any key developments in the case. “We have every confidence as a family that when the world has heard the full evidence that this will prove to be a terrible and tragic accident which has changed many lives forever. We are praying for everyone touched by this tragedy.” Tweet with a location You can add location information to your Tweets, such as your city or precise location, from the web and via third-party applications. You always have the option to delete your Tweet location history. Learn more | Summary: The last time Oscar Pistorius tweeted was Feb. 12, 2013, two days before he shot and killed girlfriend Reeva Steenkamp in what he maintains was a tragic accident in which he mistook her for an intruder. Today brought a new tweet: "A few words from my heart on oscarpistorius.com." There, the double-amputee Olympian posted a brief statement. It reads in part: "No words can adequately capture my feelings about the devastating accident" that "consumes me with sorrow. The loss of Reeva and the complete trauma of that day, I will carry with me for the rest of my life." Meanwhile, Steenkamp's parents, Barry and June, released a statement of their own. "All we are looking for is closure and to know that our daughter did not suffer on that tragic Valentine's Day," they said, per the Guardian. Steenkamp's uncle noted a private family gathering would be held today in Cape Town to mark the anniversary. "We will release balloons in her memory," he told the AFP. "They will be red and white, her favorite colors." Steenkamp's parents also said a foundation, possibly focusing on women's abuse issues, would be set up in their daughter's honor once Pistorius' murder trial is over. It begins March 3. | 874 | 291 | multi_news | en |
Summarize: Timothée Chalamet eredita da Johnny Deep un altro ruolo iconico sul grande schermo. Dopo essersi calato nei panni di Edward mani di forbice, anche se solo per uno spot ideato per il SuperBowl, l'attore 25enne candidato agli Oscar con Chiamami col tuo nome di Luca Guadagnino, sarà Willy Wonka in un film musical che racconta l'infanzia dello stravagante cioccolataio nato dalla mente di Roald Dahl. "Wonka", questo il titolo del film la cui uscita è prevista a marzo 2023, non porterà in scena una nuova versione della storia già narrata, ma farà luce sulle avventure pregresse del protagonista che lo hanno portato ad aprire la fabbrica di cioccolato più famosa al mondo, con migliaia di bizzarri Umpa Lumpa alle sue dipendenze. Timothée Chalamet prende in carico l'eredità di Gene Wilder, protagonista della storica versione del film ispirato al romanzo del 1971 ‘Willy Wonka e la fabbrica di cioccolato' e quella più recente di Johnny Deep, che nel 2005 arrivò ad incassare 475 milioni di dollari in tutto il mondo. Ma attorno all'iconico cioccolataio ruotano altri nuovi progetti per il piccolo schermo, ovvero un paio di serie animate per Netflix alle quali Taika Waititi (regista premio Oscar per Jojo Rabbit) sta lavorando: una ispirata al mondo di ‘Charlie e la Fabbrica di Cioccolato' e una sulla vita degli ‘Umpa Lumpa'. | Summary: Si intitola "Wonka" il nuovo film musical prodotto dalla Warner Bros, che avrà come protagonista il candidato premio Oscar Timothée Chalamet nei panni dello stravagante cioccolataio. Il film, la cui uscita è prevista nell'aprile 2023, non sarà una nuova versione della storia già conosciuta, ma farà luce sulle avventure pregresse e sull'infanzia di Willy Wonka, raccontando la storia che lo ha condotto ad aprire la fabbrica di cioccolato più famosa al mondo. | 335 | 110 | fanpage | it |
Summarize: Background As the central human resources agency for the federal government, OPM is tasked with ensuring that the government has an effective civilian workforce. To carry out this mission, OPM delivers human resources products and services including policies and procedures for recruiting and hiring, provides health and training benefit programs, and administers the retirement program for federal employees. According to the agency, approximately 2.7 million active federal employees and nearly 2.5 million retired federal employees rely on its services. The agency’s March 2008 analysis of federal employment retirement data estimates that nearly 1 million active federal employees will be eligible to retire and almost 600,000 will most likely retire by 2016. According to OPM, the retirement program serves current and former federal employees by providing (1) tools and options for retirement planning and (2) retirement compensation. Two defined-benefit retirement plans that provide retirement, disability, and survivor benefits to federal employees are administered by the agency. The first plan, the Civil Service Retirement System (CSRS), provides retirement benefits for most federal employees hired before 1984. The second plan, the Federal Employees Retirement System (FERS), covers most employees hired in or after 1984 and provides benefits that include Social Security and a defined contribution system. Federal Employee Retirement Application Processing Is Complex OPM and employing agencies’ human resources and payroll offices are responsible for processing federal employees’ retirement applications. The process begins when an employee submits a paper retirement application to his or her employer’s human resources office and is completed when the individual begins receiving regular monthly benefit payments (as illustrated in fig. 1). Once an employee submits an application, the human resources office provides retirement counseling services to the employee and augments the retirement application with additional paperwork, such as a separation form that finalizes the date the employee will retire. Then the agency provides the retirement package to the employee’s payroll office. After the employee separates for retirement, the payroll office is responsible for reviewing the documents for correct signatures and information, making sure that all required forms have been submitted, and adding any additional paperwork that will be necessary for processing the retirement package. Once the payroll office has finalized the paperwork, the retirement package is mailed to OPM to continue the retirement process. Payroll offices are required to submit the package to OPM within 30 days of the retiree’s separation date. Upon receipt of the retirement package, OPM calculates an interim payment based on information provided by the employing agency. The interim payments are partial payments that typically provide retirees with 80 percent of the total monthly benefit they will eventually receive.then starts the process of analyzing the retirement application and associated paperwork to determine the total monthly benefit amount to which the retiree is entitled. This process includes collecting additional information from the employing agency’s human resources and payroll offices or from the retiree to ensure that all necessary data are available before calculating benefits. After OPM completes its review and authorizes payment, the retiree begins receiving 100 percent of the monthly retirement benefit payments. OPM then stores the paper retirement folder at the Retirement Operations Center in Boyers, Pennsylvania. The agency recently reported that the average time to process a new retirement claim is 156 days. According to the Deputy Associate Director for the Center of Retirement and Insurance Services, about 200 employees are directly involved in processing the approximately 100,000 retirement applications OPM receives annually. Retirement processing includes functions such as determining retirement eligibility, inputting data into benefit calculators, and providing customer service. The agency uses over 500 different procedures, laws, and regulations, which are documented on the agency’s internal website, to process retirement applications. For example, the site contains memorandums that outline new procedures for handling special retirement applications, such as those for disability or court orders. Further, OPM’s retirement processing involves the use of over 80 information systems that have approximately 400 interfaces with other internal and external systems. For instance, 26 internal systems interface with the Department of the Treasury to provide, among other things, information regarding the total amount of benefit payments to which an employee is entitled. OPM has reported that a greater retirement processing workload is expected due to an anticipated increase in the number of retirement applications over the next decade, although current retirement processing operations are at full capacity. Further, the agency has identified several factors that limit its ability to process retirement benefits in an efficient and timely manner. Specifically, OPM noted that: current processes are paper-based and manually intensive, resulting in a higher number of errors and delays in providing benefit payments; the high costs, limited capabilities, and other problems with the existing information systems and processes pose increasing risks to the accuracy of benefit payments; current manual capabilities restrict customer service; federal employees have limited access to retirement records, making planning for retirement difficult; and attracting qualified personnel to operate and maintain the antiquated retirement systems, which have about 3 million lines of custom programming, is challenging. OPM Has a Long History of Unsuccessful Retirement Modernization Initiatives Recognizing the need to modernize its retirement processing, in the late 1980s OPM began initiatives that were aimed at automating its antiquated paper-based processes. Initial modernization visions called for developing an integrated system and automated processes to provide prompt and complete benefit payments. However, following attempts over more than two decades, the agency has not yet been successful in achieving the modernized retirement system that it envisioned. In early 1987, OPM began a program called the FERS Automated Processing System. However, after 8 years of planning, the agency decided to reevaluate the program, and the Office of Management and Budget requested an independent review of the program that identified various management weaknesses. The independent review suggested areas for improvement and recommended terminating the program if immediate action was not taken. In mid-1996, OPM terminated the program. In 1997, OPM began planning a second modernization initiative, called the Retirement Systems Modernization (RSM) program. The agency originally intended to structure the program as an acquisition of commercially available hardware and software that would be modified in- house to meet its needs. From 1997 to 2001, OPM developed plans and analyses and began developing business and security requirements for the program. However, in June 2001, it decided to change the direction of the retirement modernization initiative. In late 2001, retaining the name RSM, the agency embarked upon its third initiative to modernize the retirement process and examined the possibility of privately sourced technologies and tools. Toward this end, the agency determined that contracting was a viable alternative and, in 2006, awarded three contracts for the automation of the retirement process, to include the conversion of paper records to electronic files and consulting services to redesign its retirement operations. In February 2008, OPM renamed the program RetireEZ and deployed an automated retirement processing system. However, by May 2008 the agency determined that the system was not working as expected and suspended system operation. In October 2008, after 5 months of attempting to address quality issues, the agency terminated the contract for the system. In November 2008, OPM began restructuring the program and reported that its efforts to modernize retirement processing would continue. However, after several years of trying to revitalize the program, the agency terminated the retirement system modernization in February 2011. IT Management Weaknesses Have Contributed to OPM’s Unsuccessful Retirement Modernization Efforts OPM’s efforts to modernize its retirement system have been hindered by weaknesses in several key IT management disciplines. Our experience with major modernization initiatives has shown that having sound management capabilities is essential to achieving successful outcomes. Among others, these capabilities include project management, risk management, organizational change management, system testing, cost estimating, progress reporting, planning, and oversight. However, we found that many of the capabilities in these areas were not sufficiently developed. For example, in reporting on RSM in February 2005, we noted weaknesses in key management capabilities, such as project management, risk management, and organizational change management. Project management is the process for planning and managing all project-related activities, including defining how project components are interrelated. Effective project management allows the performance, cost, and schedule of the overall project to be measured and controlled in comparison to planned objectives. Although OPM had defined major retirement modernization project components, it had not defined the dependencies among them. Specifically, the agency had not identified critical tasks and their impact on the completion of other tasks. By not identifying critical dependencies among retirement modernization components, OPM increased the risk that unforeseen delays in one activity could hinder progress in other activities. Risk management entails identifying potential problems before they occur. Risks should be identified as early as possible, analyzed, mitigated, and tracked to closure. OPM officials acknowledged that they did not have a process for identifying and tracking retirement modernization project risks and mitigation strategies on a regular basis but stated that the agency’s project management consultant would assist it in implementing a risk management process. Without such a process, OPM did not have a mechanism to address potential problems that could adversely impact the cost, schedule, and quality of the retirement modernization project. Organizational change management includes preparing users for the changes to how their work will be performed as a result of a new system implementation. Effective organizational change management includes plans to prepare users for impacts the new system might have on their roles and responsibilities, and a process to manage those changes. Although OPM officials stated that change management posed a substantial challenge to the success of retirement modernization, they had not developed a detailed plan to help users transition to different job responsibilities. Without having and implementing such a plan, confusion about user roles and responsibilities could have hindered effective implementation of new retirement systems. We recommended that the Director of OPM ensure that the retirement modernization program office expeditiously establish processes for effective project management, risk management, and organizational change management. In response, the agency initiated steps toward establishing management processes for retirement modernization and demonstrated activities to address our recommendations. We subsequently reported on OPM’s retirement modernization again in January 2008, as the agency was on the verge of deploying a new automated retirement processing system. We noted weaknesses in additional key management capabilities, including system testing, cost estimating, and progress reporting. Effective testing is an essential activity of any project that includes system development. Generally, the purpose of testing is to identify defects or problems in meeting defined system requirements or satisfying system user needs. At the time of our review, 1 month before OPM planned to deploy a major system component, test results showed that the component had not performed as intended. We warned that until actual test results indicated improvement in the system, OPM risked deploying technology that would not accurately calculate retirement benefits. Although the agency planned to perform additional tests to verify that the system would work as intended, the schedule for conducting these tests became compressed from 5 months to 2-1/2 months, with several tests to be performed concurrently rather than in sequence. The agency identified a lack of testing resources, including the availability of subject matter experts, and the need for further system development, as contributing to the delay of planned tests and the need for concurrent testing. The high degree of concurrent testing that OPM planned to meet its February 2008 deployment schedule increased the risk that the agency would not have the resources or time to verify that the planned system worked as expected. Cost estimating represents the identification of individual project cost elements, using established methods and valid data to estimate future costs. The establishment of a reliable cost estimate is important for developing a project budget and having a sound basis for measuring performance, including comparing the actual and planned costs of project activities. Although OPM developed a retirement modernization cost estimate, the estimate was not supported by the documentation that is fundamental to a reliable cost estimate. Without a reliable cost estimate, OPM did not have a sound basis for formulating retirement modernization budgets or for developing the cost baseline that is necessary for measuring and predicting project performance. Earned value management (EVM) is a tool for measuring program progress by comparing the value of work accomplished with the amount of work expected to be accomplished. Fundamental to reliable EVM is the development of a baseline against which variances are calculated. OPM used EVM to measure and report monthly performance of the retirement modernization system. The reported results provided a favorable view of project performance over time because the variances indicated the project was progressing almost exactly as planned. However, this view of project performance was not reliable because the baseline on which it was based did not reflect the full scope of the project, had not been validated, and was unstable (i.e., subject to frequent changes). This EVM approach in effect ensured that material variances from planned project performance would not be identified and that the state of the project would not be reliably reported. We recommended that the Director of OPM address these deficiencies by, among other things, conducting effective system tests prior to system deployment, in addition to improving program cost estimation and progress reporting. In response to our report, OPM stated that it concurred with our recommendations and stated that it would take steps to address the weakness we identified. Nevertheless, OPM deployed a limited initial version of the modernized retirement system in February 2008. After unsuccessful efforts to address system quality issues, the agency suspended system operation, terminated the system contract, and began restructuring the modernization effort. In April 2009, we again reported on OPM’s retirement modernization, noting that the agency still remained far from achieving the modernized retirement processing capabilities that it had planned. Specifically, we noted that significant weaknesses continued to exist in three key management areas that we had previously identified—cost estimating, progress reporting, and testing—while also noting two additional weaknesses related to planning and oversight. Despite agreeing with our January 2008 recommendation that OPM develop a revised retirement modernization cost estimate, the agency had not completed initial steps for developing a new cost estimate by the time we reported again in April 2009. At that time, we reported that the agency had not yet fully defined the estimate’s purpose, developed an estimating plan, or defined the project’s characteristics. By not completing these steps, OPM increased the risk that it would produce an unreliable estimate and not have a sound basis for measuring project performance and formulating retirement modernization budgets. Although it agreed with our January 2008 recommendation to establish a basis for effective EVM, OPM had not completed key steps as of the time of our April 2009 report. Specifically, despite planning to begin reporting on the retirement project’s progress using EVM, the agency was not prepared to do so because initial steps, including the development of a reliable cost estimate and the validation of a baseline, had not been completed. Engaging in EVM reporting without first performing these fundamental steps could have again rendered the agency’s assessments unreliable. As previously discussed, effective testing is an essential component of any project that includes developing systems. To be effectively managed, testing should be planned and conducted in a structured and disciplined fashion. Beginning the test planning process in the early stages of a project life cycle can reduce rework later. Early test planning in coordination with requirements development can provide major benefits. For example, planning for test activities during the development of requirements may reduce the number of defects identified later and the costs related to requirements rework or change requests. OPM’s need to compress its testing schedule and conduct tests concurrently, as we reported in January 2008, illustrates the importance of planning test activities early in a project’s life cycle. However, at the time of our April 2009 report, the agency had not begun to plan test activities in coordination with developing its requirements for the system it was planning at that time. Consequently, OPM increased the risk that it would again deploy a system that did not satisfy user expectations and meet requirements. Project management principles and effective practices emphasize the importance of having a plan that, among other things, incorporates all the critical areas of system development and is to be used as a means of determining what needs to be done, by whom, and when. Although OPM had developed a variety of informal documents and briefing slides that described retirement modernization activities, the agency did not have a complete plan that described how the program would proceed in the wake of its decision to terminate the system contract. As a result, we concluded that until the agency completed and used a plan that could guide its efforts, it would not be properly positioned to move forward with its restructured retirement modernization initiative. Office of Management and Budget and GAO guidance call for agencies to ensure effective oversight of IT projects throughout all life- cycle phases. Critical to effective oversight are investment management boards made up of key executives who regularly track the progress of IT projects such as system acquisitions or modernizations. OPM’s Investment Review Board was established to ensure that major investments are on track by reviewing their progress and determining appropriate actions when investments encounter challenges. Despite meeting regularly and being provided with information that indicated problems with the retirement modernization, the board did not ensure that retirement modernization investments were on track, nor did it determine appropriate actions for course correction when needed. For example, from January 2007 to August 2008, the board met and was presented with reports that described problems the retirement modernization program was facing, such as the lack of an integrated master schedule and earned value data that did not reflect the “reality or current status” of the program. However, meeting minutes indicated that no discussion or action was taken to address these problems. According to a member of the board, OPM guidance regarding how the board is to communicate recommendations and needed corrective actions for investments it is responsible for overseeing had not been established. Without a fully functioning oversight body, OPM could not monitor the retirement modernization and make the course corrections that effective boards are intended to provide. Our April 2009 report made new recommendations that OPM address the weaknesses in the retirement modernization project that we identified. Although the agency began taking steps to address them, the recommendations were overtaken by the agency’s decision in February 2011 to terminate the retirement modernization project. OPM Has a New Plan to Improve Retirement Processing In mid-January 2012, OPM released a new plan that describes the agency’s intention to improve retirement processing through actions that include hiring and training 56 new staff to adjudicate retirement claims and 20 additional staff to support the claims process; establishing higher production standards and identifying potential retirement process improvements; and working with other agencies to improve the accuracy and completeness of the data they provide to OPM for use in retirement processing. Additionally, OPM’s new plan identifies existing and planned IT improvements to support the retirement process. Recognizing that its previous, large-scale efforts to automate the retirement process have failed, the agency characterizes its new plan as representing partial, progressive IT improvements. These efforts include providing retirees with the capability to access and update their accounts to change addresses, banking information, and tax exemptions and planning to automate retirement applications and to automatically collect retirement data from agencies’ payroll processing centers. Under this approach, OPM expects to eliminate the agency’s retirement processing backlog within 18 months and to accurately process 90 percent of its cases within 60 days. However, this goal represents a substantial reduction from the agency’s fiscal year 2009 retirement modernization goal to accurately process 99 percent of cases within 30 days. Moreover, the plan does not describe whether or how the agency intends to modify or decommission the over 80 legacy systems that support retirement processing. In summary, despite OPM’s recognition of the need to improve the timeliness and accuracy of retirement processing, the agency has thus far been unsuccessful in several attempts to develop the capabilities it has long sought. For over two decades, the agency’s retirement modernization efforts have been plagued by weaknesses in management capabilities that are critical to the success of such endeavors. Among the management disciplines the agency has struggled with are project management, risk management, organizational change management, cost estimating, system testing, progress reporting, planning, and oversight. Although the agency is now considering modest, incremental efforts to improve retirement processing, the development and institutionalization of the aforementioned management capabilities remain important to OPM’s success in improving the delivery of retirement services. Mr. Chairman, this concludes my statement today. I would be pleased to answer any questions that you or other members of the Subcommittee may have. Contact and Acknowledgments If you have any questions concerning this statement, please contact Valerie C. Melvin, Director, Information Management and Technology Resources Issues, at (202) 512-6304 or melvinv@gao.gov. Other individuals who made key contributions include Mark T. Bird, Assistant Director; Barbarol J. James; Lee A. McCracken; Teresa M. Neven; and Robert L. Williams, Jr. This is a work of the U.S. government and is not subject to copyright protection in the United States. The published product may be reproduced and distributed in its entirety without further permission from GAO. However, because this work may contain copyrighted images or other material, permission from the copyright holder may be necessary if you wish to reproduce this material separately. | Summary: The Office of Personnel Management (OPM) is the central human resources agency for the federal government and, as such, is responsible for ensuring that the government has an effective civilian workforce. As part of its mission, OPM defines recruiting and hiring processes and procedures; provides federal employees with various benefits, such as health benefits; and administers the retirement program for federal employees. OPMs use of information technology (IT) is critical in carrying out its responsibilities; in fiscal year 2011 the agency invested $79 million in IT systems and services. For over 2 decades, OPM has been attempting to modernize its federal employeeretirement process by automating paper-based processes and replacing antiquated information systems. However, these efforts have been unsuccessful, and OPM canceled its most recent large-scale retirement modernization effort in February 2011. GAO was asked to summarize its work on challenges OPM has faced in attempting to modernize the federal employee retirement process. To do this, GAO relied on previously published work in addition to reviewing OPMs recent plan for retirement services. In a series of reviews, GAO found that OPMs retirement modernization efforts were hindered by weaknesses in key management practices that are essential to successful IT modernization projects. For example, in 2005, GAO made recommendations to address weaknesses in the following areas: Project management: While OPM had defined major components of its retirement modernization effort, it had not identified the dependencies among them, increasing the risk that delays in one activity could have unforeseen impacts on the progress of others. Risk management: OPM did not have a process for identifying and tracking project risks and mitigation strategies on a regular basis. Thus, OPM lacked a mechanism to address potential problems that could adversely impact the cost, schedule, and quality of the modernization effort. Organizational change management: OPM had not adequately prepared its staff for changes to job responsibilities resulting from the modernization by developing a detailed transition plan. This could lead to confusion about roles and responsibilities and hinder effective system implementation. In 2008, as OPM was on the verge of deploying an automated retirement processing system, GAO reported deficiencies in and made recommendations to address additional management capabilities: Testing: The results of tests 1 month prior to the deployment of a major system component revealed that it had not performed as intended. These defects, along with a compressed testing schedule, increased the risk that the system would not work as intended upon deployment. Cost estimating: The cost estimate OPM developed was not fully reliable. This meant that the agency did not have a sound basis for formulating budgets or developing a program baseline. Progress reporting: The baseline against which OPM was measuring the progress of the program did not reflect the full scope of the project; this increased the risk that variances from planned performance would not be detected. In 2009, GAO reported that OPM continued to have deficiencies in its cost estimating, progress reporting, and testing practices and made recommendations to address these deficiencies, as well as additional weaknesses in the planning and oversight of the modernization effort. OPM agreed with these recommendations and began to address them, but the agency terminated the modernization effort in February 2011. More recently, in January 2012, OPM released a new plan to improve retirement processing that aims at targeted, incremental improvements rather than a largescale modernization. Specifically, OPM plans to hire new claims-processing staff, take steps to identify potential process improvements, and work with other agencies to improve data quality. Further, OPM intends to make IT improvements that allow retirees to access and update their accounts and automate the retirement application process. However, the plan reflects a less ambitious retirement processing timeliness goal and does not address improving or eliminating the legacy systems that support retirement processing. | 4,688 | 844 | gov_report | en |
Write a title and summarize: Ein Bauchredner, gelegentlich latinisierend als Ventriloquist (von lateinisch venter,Bauch' und loqui,reden') bezeichnet, manipuliert seine Stimme in einer Art, dass sie von einer anderen Person, einer Puppe oder aus einer anderen Richtung zu kommen scheint. Häufig wird die Kunst zur Unterhaltung der Menschen eingesetzt. == Lautbildung Worte ohne Bewegung des Mundes hervorzubringen ist eine Kunst. Früher glaubte man tatsächlich, die Stimme komme aus dem Bauch. Tatsächlich gibt es verschiedene Techniken des Bauchredens. Keine von ihnen hat etwas mit dem "Reden aus dem Bauch" zu tun. Entgegen der allgemeinen Meinung kann jeder das Bauchreden lernen, da kein besonderer Körperbau vonnöten ist. Man unterscheidet beim Erlernen des Bauchredens "Kieferlaute" und "Lippenlaute". Kieferlaute sind Buchstaben des Alphabets, die nicht mit den Lippen gebildet werden müssen (a, e, i, o, u, l, s...). Beim Üben muss lediglich der Kiefer still gehalten werden. Lippenlaute sind schwieriger zu bilden (b, p, f, m, w). Sie ohne den Einsatz der Lippen zu bilden, erfordert Übung. Hilfsmittel hierbei sind die Zunge und der Gaumen. Manche Bauchredner behelfen sich, == Geschichte und bekannte Bauchredner Die heute häufigste Form des Bauchredens, die in Varietes - früher auch im Vaudeville-Theater - aufgeführt wird, ist in dieser Form aber gerade mal etwas über 100 Jahre alt. Im Vaudeville lag nicht der Humor im Vordergrund eines Bauchrednerstückes, sondern die Fähigkeiten des Künstlers, sein Publikum mit schnell wechselnden Stimmen bei möglichst vielen Personen oder Puppen oder auch Tieren zu täuschen. Bekannter Bauchredner des Altertums war Eurykles von Athen. Im Mittelalter und der Frühen Neuzeit wurden Bauchredner teilweise als Zauberer oder Hexen verbrannt, wurden aber auch von Monarchen als Hofnarren eingesetzt. In den USA war Jules Vernon ein berühmter Bauchredner im Vaudeville, der seine Fähigkeiten an mehreren Figuren demonstrierte. Der vielleicht berühmteste war The Great Lester, der nur eine einzige Puppe benutzte, genannt Frank Byron, Jr., und der mit ihr viele Standards einführte, die noch heute in Vorstellungen aufgeführt werden. In Europa trat Anfang des 20. Jahrhunderts der Bauchredner Fedor Wittkowski unter dem Künstlernamen Henry Rox nicht nur mit seiner Puppe Max in Varietes auf, er dressierte auch Hunde, die er verkleidete (unter anderem als Seemann) und sprechen ließ. In der Mitte des 20. Jahrhunderts wurde ein Schüler von The Great Lester, Edgar Bergen, mit seiner Puppe Charlie McCarthy nicht nur in Varietes außerordentlich populär, er trat mit ihr sogar in Radio-Shows auf, wo man die Puppe gar nicht sehen konnte. In diesen Shows lieferte sich die Puppe, also eigentlich Bergen, u. a. Wortgefechte mit dem Schauspieler W. C. Fields. In den 1950er und 1960er Jahren wurde in den Vereinigten Staaten Paul Winchell mit seinen Puppen im Fernsehen sehr bekannt. Der bekannteste englischsprachige Bauchredner der letzten Jahre ist Jeff Dunham, während Sascha Grammel und Sebastian Reich durch ihre Fernsehauftritte die Bauchrednerkunst in Deutschland wieder mehr in das Rampenlicht gerückt haben. Grosse Popularität - auch ausserhalb seines Heimatlandes - erlangte in den 1970er und 1980er Jahren der Schweizer Urs Kliby mit seiner vorwitzigen Eselpuppe Caroline. == Sonstiges In den Batman-Comics tritt der Schurke Arnold Wesker als Bauchredner (englisch Ventriloquist) auf. | Title: Bauchredner Summary: Ein Bauchredner unterhält die Menschen. Dabei spricht der Bauchredner so, dass man nicht erkennt, wie er spricht. Bauchreden hat mit dem Bauch eigentlich nichts zu tun. Der Künstler spricht in Wirklichkeit mit dem Mund. Die meisten Bauchredner haben eine Puppe, die sie gleichzeitig bedienen. So sieht es aus, als wenn sie sich mit der Puppe unterhalten würden. Bauchreden macht man heute aus Spass und zur Unterhaltung. Früher haben Bauchredner so getan, als wenn sie mit verstorbenen Menschen sprechen würden. == Woher kommt das Bauchreden? Schon im Altertum gab es Bauchredner, zum Beispiel bei den Alten Griechen. Einer davon war Eurykles von Athen, der vor 2400 Jahren lebte. Angeblich befanden sich in seinem Bauch Dämonen, die durch ihn gesprochen haben. Ähnliche Geschichten kennt man auch von Völkern in Afrika. Viel später, in den Jahren nach 1700, wurde Bauchreden eine Show. In Theatern und auf Jahrmärkten taten die Künstler so, als käme eine Stimme aus der weiten Ferne her. Oder unterhielten sich mit einem unsichtbaren Gast. Einige Künstler arbeiteten auch schon mit Puppen. Später traten Bauchredner im Variete auf, ähnlich wie Zauberkünstler. == Wie redet man so? Jeder kann lernen, so zu reden, dass von aussen kaum erkennbar ist, dass man redet. Viele Laute bilden wir im Mund, zum Beispiel die Selbstlaute a, e, i, o und u. Man muss lernen, den Mund ein wenig offen zu halten, damit die Luft entweichen kann. Das versteckt man vielleicht unter einem Lächeln. Man kann üben zu reden, ohne dass man den Kiefer bewegt. Mit einem Halstuch kann man da noch manche Bewegung verbergen. Wirklich schwierig ist es, bestimmte Laute zu sprechen, für die wir beide Lippen brauchen, wie das b und das p. Zum Teil kann man versuchen, die untere Lippe durch die Zunge zu ersetzen. Dabei kommt aber eher eine Art d und t heraus. Es macht jedoch gar nicht so viel aus, wenn man einige Laute etwas undeutlich spricht: Die Zuschauer können das meiste dennoch verstehen. Bauchredner sagen oft nicht einfach etwas, das ihnen so einfällt. Sie haben genau überlegt und auswendig gelernt, was sie selbst sagen werden und was die Puppe spricht. So kann man zum Beispiel verhindern, dass die Puppe viele schwierige Laute sprechen muss. Der Künstler sagt etwa: "Magst du deine neue Barbiepuppe?", und die Puppe antwortet nur: "Ja, genau, die finde ich richtig gut." == Was muss ein Bauchredner noch können? Für die Zuschauer ist es nicht interessant genug, wenn jemand "bauchredet". Sie möchten gut unterhalten werden, die Show soll lustig sein. Der Bauchredner muss sich also witzige Dinge ausdenken, die sich für ein Gespräch von zwei Leuten eignen. Überhaupt muss der Bauchredner ein richtiger Puppenspieler sein. Während er spricht, muss sich die Puppe bewegen, so, als wenn sie leben würde. Ausserdem ist er selbst Teil der Show und muss zum Beispiel mit der Puppe umgehen wie mit einem anderen Menschen, dem er zuhört. | 807 | 685 | klexikon_de | de |
Summarize: STATEMENT OF GOVERNMENT INTEREST The invention described herein may be manufactured and used by or for the Government for governmental purposes without the payment of any royalty thereon. BACKGROUND OF THE INVENTION This invention relates to a container holder assembly and, more particularly, to a bottle holder assembly, adapted for use as an inflight holder of an intravenous bottle. Presently there is no simple, satisfactory method of (or an apparatus for) suspending intravenous bottles or other hanging equipment at patients' litters while caring for litter patients during aeromedical airlift missions. The current method, which is far from being completely satisfactory, is to secure these articles with adhesive tape and gauze bandages, which at best is makeshift. Obviously, therefore, there is a dire need for a holder, especially of an intravenous bottle, in caring for patients at their litters in the aircraft. I have invented such a holder; and, by fulfilling this need, I have significantly advanced the state-of-the-art. SUMMARY OF THE INVENTION An object of this invention is to provide a container holder assembly that can be used at a patient's litter in an aircraft. Another object of this invention is to provide such a container holder assembly which is relatively inexpensive to manufacture and also is quickly attachable, easily adjustable, compact, and lightweight. Still another object of this invention is to teach a specific preferred embodiment of the hereinabove described container holder assembly, wherein the preferred embodiment is especially adapted for use as an inflight holder of an intravenous bottle. These objects, and other equally important and related objects, of this invention will become readily apparent after a consideration of the description of the invention, coupled with reference to the Figures of the drawings. DESCRIPTION OF THE DRAWINGS FIG. 1 is a pictorial view, in perspective, of the preferred embodiment of the invention in use in its working environment; FIG. 2 is a pictorial view, in perspective, of the preferred embodiment of the invention in its working environment, although not in use; and FIGS. 3, 4 and 5 are various views of the same major component of the preferred embodiment of the invention. DESCRIPTION OF THE PREFERRED EMBODIMENT With reference to FIG. 1, therein is shown a preferred embodiment 10 of my inventive container holder assembly, as adapted for use in an inflight holder of an intravenous bottle 100, in its working environment in a multimission aircraft, during an aeromedical airlift operation, wherein said aircraft has a plurality of litters (i.e., medical stretchers), such as representative one 200 shown in partially fractured form, each of which said litters has (but is not limited to) a side carrying pole 201 and a canvas, or the like, bed member 202. The litters are disposed in spaced-apart horizontal position and relationship, and are also "stacked" one above the other in vertical spaced-apart position and relationship. The aircraft also has therein a plurality of litter supports, such as representative one 300, each of which includes (but is not limited to): a vertically disposed support strap 301, conventionally made of webbed material, secured at its upper end to the interior of the aircraft; and, a carrying pole holder 302, conventionally a clasp or clamp, which also holds the vertically disposed support strap 301. It is to be assumed, for explanatory purposes, that there is a patient in a litter vertically below representative litter 200, which said patient is to be fed, or otherwise medicated, intravenously using preferred embodiment 10 as the inflight holder of a conventional intravenous bottle, such as 100, which has two ends 101 and 102, with a neck 103 and an outlet 104 at one end 101, and a bottom 105 and a bale (i.e., a bail wire) 106 at the other end 102. It is to be noted that the intravenous bottle 100 is inverted when in use. Still with reference to FIG. 1, the preferred embodiment 10 includes: a cradle (or bracket) 11 generally configurated in the form of an inverted T, wherein the T has a vertical leg 12 with an upper end 13 and a lower end 14, and a horizontal leg 15, and wherein the cradle 11 is suitably configurated and dimensioned so as to be removably attachable to, and slideably movable vertically along, any one of the litter support straps, such as representative one 301; a hook 16 that is removably connected to the upper end 13 of the vertical leg 12 of the inverted T-shaped cradle, with the hook 16 suitably configurated and dimensioned to accept and to hold the bale (i.e., the bale wire) 106 of the intravenous bottle 100 when the bottle 100 is in the inverted operative position; and, a strap (or harness) subassembly, generally designated 20, that is suitably configurated and dimensioned to accept, and to assist in holding and in supporting, the inverted intravenous bottle 100 at the neck 103 and outlet 104 end 101. Again with reference to FIG. 1, the cradle 11 has a first slot 13A in the upper end 13 of the vertical leg 12 thereof, and has a second slot 14A in the lower end 14 of the vertical leg 12 thereof. Additionally, each slotted end 13A and 14A is bent backwardly, as indicated by the respective arrows, to engage with, and to frictionally hold, the litter support strap 301. Further, the upper slotted end 13 preferably has, but need not have, an upwardly extending flange 13B which assists in preventing accidental lateral slippage of the support strap 301 out of upper slot 13A. Also, the lower slotted end 14 preferably has, but need not have, a forwardly extending flange 14B also which assists in preventing accidental lateral slippage of support strap 301 out of lower slot 14A. Both the cradle 11 and the hook 16 are preferably made of metal, with the cradle 11 preferably made of aluminum as a first preference and of stainless steel as a second preference, and with the hook 16 preferably made of stainless steel. The strap or harness subassembly includes, but is not necessarily limited to: an upper strap 21 having a first end portion 22A and a second end portion 22B, with these portions releasably connectable to each other by suitable means; an intermediate strap 23 also having a first end portion 24A and a second end portion, with these end portions attached to the upper strap 21 at, respectively, different locations thereof 21; and, a first lower strap 25 and a second lower strap 26, wherein each lower strap 25 and 26 has two end portions, with one end portion of each lower strap attached to the intermediate strap 24 at different locations, and with each of the respective other end portions releasably connectable to each other by suitable means. With reference to FIG. 2, therein is shown the preferred embodiment 10 of my inventive bottle holder assembly in its working environment, as explained hereinbefore, but not in use, and not holding and supporting the intravenous bottle 100, FIG. 1. In this view the preferred structural details of the horizontal leg 15 of the cradle 11, and of the strap subassembly 20, can be easily seen. As to the horizontal leg 15, there is a plurality of openings (in this case two of them) therein, such as representative ones 15A and 15B, to accept and to hold the strap subassembly 20 generally, and the upper strap 21 specifically. Additionally, the horizontal leg 15 has a first end area 15C and a second end area 15D which preferably are, but need not be, bent forwardly (as indicated by directional arrows) to conform to the external surface of the intravenous bottle 100, FIG. 1. As to the strap (or harness) subassembly 20, easily seen is upper strap 21 with first end portion 22A and second end portion 22B; intermediate strap 23 with first end portion 24A and second end portion 24B; and, a first lower strap 25 with first end portion 26A and second end portion 26B, and a second lower strap 27 with first end portion 28A and second end portion 28B. It is to be noted intermediate strap 23, because of the attachment of end portions 24A and 24B to upper strap 21 at different locations thereof 21, becomes (i.e., forms in the shape of) a half loop which is generally designated 24C for identification purposes. It is also to be remembered: that the end portions 22A and 22B of upper strap 21 are releasably connectable to each other by suitable means; and, that second end portion 26B of lower strap 25, and first end portion 28A of second lower strap 27, are also releasably connectable to each other also by suitable means. In that regard, it is to be noted that said suitable means may comprise, solely as a matter of preference, complementary "Velcro" pieces. Of course, other suitable means may be used. It is further to be noted that each of the straps 21, 23, 25 and 27 preferable, but not necessarily, are made of nylon web material. Now, with reference to FIG. 3, therein is shown a front view of the cradle (or bracket) component 11 of the preferred embodiment 10 of my inventive bottle holder assembly, previously shown in FIGS. 1 and 2. Also clearly shown in FIG. 3 are: vertical leg 12; horizontal leg 15; vertical leg upper end 13 with slot 13A and flange 13B; vertical lower end 14; horizontal leg first end area 15C with opening 15A; and, horizontal leg second end area 15D with opening 15B. FIG. 4 is the view taken from, and seen along, line 4-4 in FIG. 3. Seen therein is vertical leg upper end 13 and flange 13B thereof. Now, with reference to FIG. 5 which is the view seen from, and taken along, line 5-5 in FIG. 3, shown therein is cradle component 11. Also shown are: vertical leg 12; horizontal leg 15; vertical leg upper end 13 with flange 13B; vertical leg lower end 14; and, horizontal leg second end area 15D with second opening 15B therein. For orientation purposes, directional arrows and appropriate legends are also shown. MANNER OF OPERATION AND/OR USE OF THE PREFERRED EMBODIMENT The manner of operation and/or use of the preferred embodiment 10 of my inventive bottle holder assembly, as adapted for use as an inflight holder of an intravenous bottle aboard an aircraft, can very easily be ascertained by a person of ordinary skill in the art, from the foregoing description, coupled with reference to the Figures of the drawings. For others, it is sufficient to say in explanation (and, with reference to FIGS. 1-5, inclusive) that to attach the bottle holder assembly 10 to support strap 301, firstly, the support strap 301, at the desired height, is slipped into slot 13A, and behind flange 13B, of upper end 13 of vertical leg 12 of cradle 11. Then, while the assembly 10 as a whole is pressed toward and against the support strap 301, the support strap 31 is slipped into slot 14A, and behind flange 14B, of lower end 14 of vertical leg 12 of cradle 11. In this regard, it is to be noted that, because the upper end 13 is bent backwardly, there is a substantial, frictional gripping action between the support strap 301 and the slot 13A in the bent upper end 13 of the vertical 12 of cradle 11. Likewise, there is a frictional gripping action between the support strap 301 and the slot 14A in the bent lower end 14. As a result, the assembly 10 as a whole is frictionally held suspended, and secured from the support strap 301. Nevertheless, the cradle is easily removable, if so desired, merely by reversing the abovementioned attachment procedure (i.e., unhooking the ends 13 and 14 of the vertical leg 12 of the cradle 11 from the support strap 301). On the other hand, if desired, the vertical leg 12 may be slideably moved upwardly or downwardly along support strap 301, without removing the cradle 11 from the support strap 301. To attach the intravenous bottle 100 to the holder 10, firstly the intravenous bottle is inverted. Then, the bottle bale 106 is slipped over the hook 16. After that, the bottle 100 is encircled with the upper strap 21, by pressing the "Velcro" tape ends 22A and 22B firmly together. Then, the bottle 100 is so positioned that the back of the neck 13 at lower end 101 of bottle 100 is on, is abutting with, and is supported by the dangling half-loop intermediate strap 23. After that, the "Velcro" tape ends 26B and 28A of, respectively, lower straps 25 and 27 are overlapped and pressed firmly, thereby completing the removable attachment of the intravenous bottle 100 to the bottle holder assembly 10. To release the bottle 100 from the holder 10, the above procedure is simply reversed. CONCLUSION It is abundantly clear from all of the foregoing, and from the Figures of the drawings herein, that the stated and desired objects of my invention have been attained. In addition, related desirable objects also have been achieved. It is to be noted that, although there have been described the fundamental and unique features of my invention as applied to a particular preferred embodiment, various other embodiments, adaptations, substitutions, additions, omissions, and the like will occur to, and can be made by, those of ordinary skill in the art, without departing from the spirit of my invention. For example, my invention may be adapted to hold and/or to suspend gastric and urinary drainage bags and oxygen and/or suction tubing. | Summary: A bottle holder assembly in a preferred embodiment adapted for use as an inflight intravenous bottle holder aboard an aircraft on an aeromedical airlift mission. The holder comprises: a cradle, generally configurated in the form of an inverted T, with an upper and a lower end and with a slot in each end, and with each end bent backwardly; a hook removably connected to the upper end; and, a strap subassembly attached to the cradle and suitably configurated and dimensioned to accept a standard intravenous bottle which is in the inverted, operative position. The intravenous bottle holder is removably attachable to, and slideably movable along, any one of the plurality of conventional litter support straps which such an aircraft is equipped. The cradle of the bottle holder is held in a gripping action with the support strap because of friction. The bottle holder is exceptionally well suited for the purpose intended, and obviates the current, unsatisfactory, use of makeshift methods to support and use the inverted intravenous bottle. | 3,512 | 229 | big_patent | en |
Summarize: Showgoers visit the Intel booth on the first day of the Consumer Electronics Show (CES) in Las Vegas January 8, 2013. DANA POINT, California Top chipmaker Intel Corp plans to launch an Internet television service this year with live and on-demand content, entering a hotly competitive race as its core PC business erodes. Shifting into an unfamiliar and potentially costly market in which Intel lacks experience and relationships, Erik Huggers, vice president and general manager of Intel Media, said he is negotiating with content providers. He said hundreds of Intel employees and their families are already testing a set-top box the company will sell as part of the service. Intel's move puts it into competition with heavyweights like Apple, Amazon and Google that believe the $100 billion cable television ecosystem is ripe for change. The chipmaker plans to offer consumers smaller bundles of content than those currently offered by cable operators, Erik Huggers, vice president and general manager of Intel Media, told the AllThingsDigital "Dive into Media" conference on Tuesday. Asked if Intel has inked any content deals, Huggers said he is working with providers and is confident Intel will have a compelling product to launch this year. "We have been working for (the past) year to set up Intel media, a new group focused on developing an Internet platform," Huggers said. "It's not a value play, it's a quality play where we'll create a superior experience for the end user." Intel has struggled to get its virtual television service off the ground due to unwillingness on the part of major media content providers to let the company unbundle and license specific networks and shows at a discount to what cable and satellite partners pay, according to sources. Silicon Valley has been taking aim at the U.S. cable television market - dominated by major distributors such as Comcast and DirecTV Group and program makers like Walt Disney Co and Time Warner Inc. Technology companies see opportunities due to reasons ranging from shifting viewer habits to mounting programming costs. A STEP BEYOND Intel's plan, if successful, would go further than products currently offered by Apple, Amazon and Netflix by offering live programming as well as on-demand content. "There is an opportunity to offer a bundle that can be curated by the consumer, an opportunity to create smarter bundles," Huggers said. Intel's set-top-box will also have a camera that could be used to automatically steer content and ads toward specific users. "There's a scenario where the TV recognizes that it's you and says 'Hey, I know what you like. I know what you want to watch', versus the environment we're in today where the TV literally is not interested in you at all," Huggers told Reuters In an interview. Some media executives are skeptical that Intel will be able to convince content providers to agree to terms that are attractive enough to make its service viable. That view was shared by Bernard Gershon, head of digital consultancy GershonMedia and a former Disney senior vice president for strategic planning who helped develop Disney's online strategy. "The chance that Intel launches is zero," Gershon, who speaks with media and digital executives, told Reuters at the conference. "They haven't cut any deals with any content companies, and they are not offering something that differentiates itself enough on service or price to get the deals done." Analysts see Intel's leap into Internet television, along with its growing focus on smartphones and tablets, as a way to diversify beyond the slowing PC market. "The question you have to ask with Intel is, Is anything they do big enough to move the needle?" said Stacy Rasgon, an analyst at Sanford Bernstein. "You're not going to make or break the company on something like this." Huggers said in the interview that Intel employees are testing the device's user interface, sound and picture quality and other features. "We're actively testing it in the field with employees. It's not the final product, but it's certainly functional," he said. Industry insiders have said Apple may unveil a TV-based device that has the potential to shake up the cozy television content and distribution industry the way the iPod and iPhone disrupted music and mobile content. Sources say Apple, which already sells a $99 set top box called Apple TV that streams Netflix and other content, has opened discussions with providers but it is unclear how much headway it has made, despite its reputation as a tough negotiator. Huggers previously worked at Microsoft and the BBC, where in 2007 he launched iPlayer, an online service letting viewers catch up with programs they missed on regular television. "The model we envision is a model where live television and catch-up television live in the same paradigm," Huggers said. Intel's shares closed up 0.76 percent, at $21.19. (Reporting By Noel Randewich and Ronald Grover; editing by Andrew Hay, Maureen Bavdek, Leslie Adler and David Gregorio) Erik Huggers, General Manager of Intel Media, has confirmed that the company is, in fact, working on providing television over the internet, and it plans to do so with new consumer hardware that Huggers described as something with "beautiful industrial design." He says the service will be relatively full-featured: "we will have live television, catch-up television, on-demand, [and] a set of applications." The hardware will also include a camera — which can be turned off — that will apparently watch users as they watch TV (including, possibly, for targeting ads). Huggers said one use case for the camera could include synchronizing viewing with viewers across the country for a "real social experience." The camera could also theoretically recognize users in order to provide personalized show recommendations. Discussing Intel's plans at the Dive into Media conference, Huggers declined to name the service, which will launch later this year. "We're working with the entire industry to figure out how to get proper television," Huggers says, pointing out that making the consumer box isn't nearly as difficult as making deals to provide the content. Pressed about whether or not Intel would offer a la carte television channels or bundles of channels, Huggers said that consumers want "choice, control, and convenience," but said that he really did "believe that there is value in bundles." He suggested that Intel would try to do bundles "right," and that "I don't believe that the industry is ready for a la carte." That apparently means creating bundles that focus on "curation" instead of "volume" — which could mean content-specific bundles. "We're working with the entire industry to figure out how to get proper television." Another challenge is building the software. Huggers repeated complaints we just heard from Facebook about the standard TV guide grid, saying "it reminds me of my first computer, the Commodore 64. I think there's a lot of room for improvement there." He also hinted that Intel would "delight" consumers with other features — referencing how difficult it is to integrate a new set-top box in a standard living room setup. He also said that changing channels takes too long on most other digital channels, something he expects Intel to solve. Huggers says that "there is no platform out there today" that could deliver the experience Intel is aiming towards, but it will be available on devices like the iPad. Intel will use the HEVC video codec instead of H.264, which Huggers says can provide much better video. Speaking to the (very real) concern of data caps on broadband, he says that Intel thinks they'll stay within them for most users — but long term those caps will either go up or go away. As for cost, Huggers said that the new service is "not about a value play," but instead offering a "vastly superior experience," which along with his comments about channel bundling suggests that Intel is not likely to save consumers much money over a more traditional cable subscription. Why is Intel doing all this? "Intel is very interested in [having] a direct connection to the consumer," Huggers says, which is a fairly big change for the chip maker. Huggers says there is very broad support for Intel Media's project within the company at large, and that the new TV service and box are more than just experiments. Even so, Intel Media is somewhat separated from the rest of the company in a separate building with a separate group — similar to what Microsoft did with the Xbox. Update: CNET spoke to Huggers after the event, and learned a few more interesting tidbits about the service, primarily that it won't be Intel-branded. "When I say Intel, you will automatically think 'Inside.' That's what we've all been trained," he told the publication. The service will also come to smartphones at some point, and the software will be constantly updated. Intel's already testing the service inside employee homes, according to the executive. Intel says it does have plans to offer its own television service this year, as rumors suggested, but it won't quite be the a la carte alternative that cord cutters have dreamed about. Yi-ting Chung / Reuters Intel says it does have plans to offer its own television service this year, as rumors suggested, but it won’t quite be the a la carte alternative that cord cutters have dreamed about. Instead, Intel wants to offer “curated” bundles of TV channels, said Erik Huggers, General Manager of Intel Media, at this week’s Dive Into Media conference. These bundles would be smaller than the packages that cable and satellite providers offer, and would allow for more customization, according to Reuters, but they won’t necessarily be cheaper. Intel plans to stream these channels over the Internet, using set-top boxes powered by the company’s own processors, and would also make the service available on other devices, such as PCs and tablets. As The Verge reports, Huggers said Intel’s devices would have a “beautiful industrial design,” and would come equipped with a camera, which could recognize individual users and recommend things to watch. In addition to streaming live television, the device will support DVR-style content, on-demand shows and apps. The idea is to have a single streaming device–and a single input on your television–that handles all your viewing needs, rather than just being a supplement like Roku or Apple TV. By combining all kinds of content into one interface, TV providers can start to move away from the traditional grid-like channel guide, and make it easier to find interesting content through recommendations or search. Many other companies have tried, or have been trying, to make this happen. Google TV, for instance, pulls in channel listings from your cable box, and indexes those shows alongside other online sources, such as Netflix and Amazon. But Google doesn’t actually offer any live television packages on its own, so Google TV can feel a bit like a hack. Microsoft has made a handful of deals to let cable subscribers watch shows on the Xbox 360, but support from TV providers has been limited so far. Apple has reportedly been trying to come up with its own TV service, similar to what Intel is putting together, but its negotiations with Hollywood have been slow-going. As the issues with those other companies have shown, software and technology are only one side of the equation. There’s also the business side, which involves convincing Hollywood that a new kind of TV service isn’t bad for their bottom lines. It seems that Intel is the only one to pull it off so far, but at a price: while rumors claimed that Intel wanted to offer a la carte programming, where you only pay for the channels you want, that’s no longer the plan. “I don’t believe that the industry is ready for a la carte,” Huggers said. We’ll have to wait and see what Intel’s curated bundles look like by comparison. Huggers said Intel will launch the service later this year under a new brand sold directly to consumers, but the company hasn’t announced a launch date or pricing. One other remark from Huggers seems worth mentioning, as reported by The Verge: “Intel is very interested in [having] a direct connection to the consumer,” Huggers said. After television, could Intel-made phones and tablets be next on the list? | Summary: Chip-maker Intel plans to venture into the world of Internet TV sometime this year. As Time explains, the company wants to offer "curated" packages of TV channels that would be streamed over the Internet. The packages would be smaller than the typical ones offered by cable and satellite companies, and thus more easily customized, but they might not be any cheaper. Company employees already are testing set-top boxes. Intriguing, maybe, but not all media executives are sold. "The chance that Intel launches is zero," one tells Reuters. "They haven't cut any deals with any content companies, and they are not offering something that differentiates itself enough on service or price to get the deals done." The Verge, meanwhile, notes an interesting facet: "The hardware will also include a camera-which can be turned off-that will apparently watch users as they watch TV (including, possibly, for targeting ads)." | 2,653 | 195 | multi_news | en |
Summarize: Seahorses are notoriously slow and awkward. But as scientists have just learned, their unique shape allows them to overcome these limitations. Despite their cute and unassuming appearance, seahorses are actually proficient killers. Seahorses are slow. Like, ridiculously slow. In fact, the researchers who conducted the study say they're slowest swimming fish known to science. But they can capture prey that swim at incredible speeds — and at an incredibly proficient rate; in ideal conditions, seahorses catch their intended prey 90% of the time, a success rate that would be amazing for any predator. So how's this possible? New research shows it all has to do with the unique shape of the seahorse's head and a technique known as "pivot" feeding — a strategy requiring a sudden, rapid movement at extremely close range. But getting close to prey is not easy underwater, as many of these tasty creatures have evolved to become hydrodynamically sensitive. An approaching animal can often trigger an escape response. Seahorse prey, namely copepods, can detect waves from incoming predators and then jolt away at speeds of more than 500 body lengths per second. That's like a human swimming at 2,000 mph (3,218 km/h)! Advertisement So, for pivot feeding to work, seahorses have evolved a rather unique morphology. Its head creates what the scientists call a reduced fluid deformation zone, or "no wake zone." Essentially, its a perfectly crafted shape, particularly above the end of the snout, that minimizes the degree to which water is disturbed by an object moving through it. This allows seahorses to approach highly sensitive copepod prey undetected. And then, using a quick and deadly strike, it's game over. To reach this conclusion, a research team from the University of Texas at Austin used a special technique to capture the movement of water around the seahorses in 3D. The technique, called holographic and particle image velocimetry (PIV), utilizes a microscope fitted with a laser and a high-speed digital camera. Read the entire study at Nature Communications: "Morphology of seahorse head hydrodynamically aids in capture of evasive prey". What does it take to catch your prey 90 percent of the time? For the seahorse, the shape of its head, along with its stealthy approach and lightning speed, literally makes a meal of some of the ocean’s most sought after prey: the copepod, according to a new study. “When you’re a copepod everything in the ocean essentially wants to eat you,” said study author Brad Gemmell, a marine biologist at the University of Texas at Austin. “They’ve evolved some pretty impressive ways to avoid being captured by predators.” This includes sensitivity and speed. Copepods, a type of small crustacean, can’t see images and rely on a remarkable sensitivity to fluid disturbances to sense predators approaching. Once they detect them, they’re outta there, roadrunner style. “They have one of the shortest response length times seen in the animal kingdom,” Gemmell said: About two or three milliseconds from the time they sense a signal to the time they scram. They can reach speeds of over 500 body lengths per second. To catch something that fast you have to be just a bit faster, and a previous study had shown that seahorses were better at getting copepods under calm conditions than other fish. Gemmell says they had a 90 percent success rate compared to the 30-40 percent success rate of other predators. (Related: “First Video of Rare Brightly Colored Seahorse.”) This remarkable lead prompted the question of why seahorses are such wicked predators, and the new study shows that the head shape of the seahorse, “creates this zone with very little disturbance, which allows them to get really close to these very sensitive, highly evasive copepods,” said Gemmell. Speedy Seahorse It might seem counterintuitive: the head of a sea horse seems bulkier than other, sleeker fish, but they have what’s called pivot or pipette method of feeding, a trait of the Syngnathid fish family which includes seahorses, pipefish, and sea dragons. The seahorse snout is narrow, elongated, and rounded on top, and since it’s quite small, it creates less disturbance in the water, allowing it to get within one millimeter of a copepod. It then sucks in prey with its mouth, which isn’t much bigger than the prey itself. It can also extend its mouth away from the thicker part of its head and swing it “forward and upward and cover the distance to the prey in about one millisecond.” (Related: “How Seahorses Evolved to Swim ‘Standing Up.’“) So even the zippy copepod’s getaway speed of two or three milliseconds is no match for the one millisecond strike of the seahorse. “Once the seahorse gets within range the story is pretty much written, as far as the fate of the copepod goes,” said Gemmell, whose study is published today in the journal Nature Communications. According to the study, large tendons of the epaxial muscles—muscles along the vertebrae—are “hypothesized to store and release energy to accelerate the head.” The researchers used high-speed digital techniques to capture the movements of the live dwarf seahorse, Hippocampus zosterae, and fluid disturbance in 3-D imagery at the strike zone. They also used preserved seahorse specimens and positioned them in flume studies, a method that allows researchers to control the flow of the water so that any disturbance would be clearly generated by the head of the animal. “The result was this zone of low disturbance is still there, even on a dead fish, so it can be done merely with the shape of the head,” added Gemmell. Calmer Seas Crucial Seahorses, Gemmell points out, are less successful in more turbulent environments where other fish have an easier time capturing copepods. The slow, deliberate stealth of the seahorse works in the calmer environments of deep water and coral reefs. The study refers to the calm space in the water that the seahorse’s head creates as a “quiet zone,” which, combined with other factors, is bad for the copepod. Guess they’re not familiar with the old stand by, “Things are quiet… a little too quiet.” Follow Liz Langley on Facebook and Twitter. | Summary: The seahorse is one of the lousiest, slowest swimmers in the ocean, and yet it manages to catch one of the fastest-swimming creatures of the deep with an astonishing success rate of 90%, reports the BBC. What gives? Thank that ingenious snout. Researchers discovered that it's shaped in such a way that it allows the seahorse to ever-so-slowly creep up on its prey without creating a telltale wake. Once it gets within a millimeter, it's game over, thanks to a quick-strike movement known as "pivot" feeding, explains i09. The shape of the head "creates this zone with very little disturbance, which allows them to get really close to these very sensitive, highly evasive copepods," one of the Texas researchers tells National Geographic. As for those copepods, they are tiny crustaceans that can pick up on the wave movements of predators and zip away at speeds that Aquaman would envy-500 body lengths per second, or the equivalent of a human swimming 2,000mph. All the more incredible that the poky seahorse "can overcome one of the most talented escape artists in the aquatic world," says the researcher. | 1,456 | 278 | multi_news | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Dam Rehabilitation and Repair Act of 2007''. SEC. 2. REHABILITATION AND REPAIR OF DEFICIENT DAMS. (a) Definitions.--Section 2 of the National Dam Safety Program Act (33 U.S.C. 467) is amended-- (1) by redesignating paragraphs (4), (5), (6), (7), (8), (9), (10), (11), (12), and (13) as paragraphs (5), (6), (7), (8), (9), (10), (12), (13), (14), and (15), respectively; (2) by striking paragraph (3) and inserting the following: ``(3) Administrator.--The term `Administrator' means the Administrator of FEMA. ``(4) Deficient dam.--The term `deficient dam' means a dam that the State within the boundaries of which the dam is located determines-- ``(A) fails to meet minimum dam safety standards of the State; and ``(B) poses an unacceptable risk to the public.''; and (3) by inserting after paragraph (10) (as redesignated by paragraph (1)) the following: ``(11) Rehabilitation.--The term `rehabilitation' means the repair, replacement, reconstruction, or removal of a dam that is carried out to meet applicable State dam safety and security standards.''. (b) Program for Rehabilitation and Repair of Deficient Dams.--The National Dam Safety Program Act is amended by inserting after section 8 (33 U.S.C. 467f) the following: ``SEC. 8A. REHABILITATION AND REPAIR OF DEFICIENT DAMS. ``(a) Establishment of Program.--The Administrator shall establish, within FEMA, a program to provide grant assistance to States for use in rehabilitation of publicly-owned deficient dams. ``(b) Award of Grants.-- ``(1) Application.--A State interested in receiving a grant under this section may submit to the Administrator an application for such grant. Applications submitted to the Administrator under this section shall be submitted at such times, be in such form, and contain such information, as the Administrator may prescribe by regulation. ``(2) In general.--Subject to the provisions of this section, the Administrator may make a grant for rehabilitation of a deficient dam to a State that submits an application for the grant in accordance with the regulations prescribed by the Administrator. The Administrator shall enter into a project grant agreement with the State to establish the terms of the grant and the project, including the amount of the grant. ``(c) Priority System.--The Administrator, in consultation with the Board, shall develop a risk-based priority system for use in identifying deficient dams for which grants may be made under this section. ``(d) Allocation of Funds.--The total amount of funds appropriated pursuant to subsection (f)(1) for a fiscal year shall be allocated for making grants under this section to States applying for such grants for that fiscal year as follows: ``(1) One-third divided equally among applying States. ``(2) Two-thirds among applying States based on the ratio that-- ``(A) the number of non-Federal publicly-owned dams that the Secretary of the Army identifies in the national inventory of dams maintained under section 6 as constituting a danger to human health and that are located within the boundaries of the State; bears to ``(B) the number of non-Federal publicly-owned dams that are so identified and that are located within the boundaries of all applying States. ``(e) Cost Sharing.--The Federal share of the cost of rehabilitation of a deficient dam for which a grant is made under this section may not exceed 65 percent of the cost of such rehabilitation. ``(f) Authorization of Appropriations.-- ``(1) In general.--There is authorized to be appropriated to carry out this section-- ``(A) $50,000,000 for fiscal year 2007; and ``(B) $100,000,000 for each of fiscal years 2008 through 2010. ``(2) Staff.--There are authorized to be appropriated to provide for the employment of such additional staff of FEMA as are necessary to carry out this section $400,000 for each of fiscal years 2007 through 2009. ``(3) Period of availability.--Sums appropriated pursuant to this section shall remain available until expended.''. (c) Conforming Amendment.--Such Act (other than section 2) is further amended by striking ``Director'' each place it appears and inserting ``Administrator''. SEC. 3. RULEMAKING. (a) Proposed Rulemaking.--Not later than 90 days after the date of enactment of this Act, the Administrator of the Federal Emergency Management Agency shall issue a notice of proposed rulemaking regarding the amendments made by section 2 of this Act. (b) Final Rule.--Not later than 120 days after the date of enactment of this Act, the Administrator shall issue a final rule regarding such amendments. | Title: To amend the National Dam Safety Program Act to establish a program to provide grant assistance to States for the rehabilitation and repair of deficient dams Summary: Dam Rehabilitation and Repair Act of 2007 - Amends the National Dam Safety Program Act to require the Administrator of the Federal Emergency Management Agency (FEMA) to establish a program to provide grant assistance to states for use in rehabilitating publicly-owned dams that fail to meet minimum safety standards and pose an unacceptable risk to the public (deficient dams). Sets forth provisions regarding procedures for grant awards and fund allocation. Requires the Administrator to develop a risk-based priority system for identifying deficient dams for which such grants may be made. Limits the federal share of rehabilitation costs to 65%. | 1,255 | 164 | billsum | en |
Write a title and summarize: SECTION 1. METALLURGICAL COAL MINING CREDIT. (a) General Rule.--Subpart D of part IV of subchapter A of chapter 1 of the Internal Revenue Code of 1986 (relating to business related credits) is amended by adding at the end thereof the following new section: ``SEC. 45A. METALLURGICAL COAL MINING CREDIT. ``(a) General Rule.--For purposes of section 38, the metallurgical coal mining credit determined under this section for the taxable year is the lesser of-- ``(1) the applicable percentage of the aggregate amount of the premiums paid or accrued by the taxpayer during the taxable year pursuant to chapter 99 of this title (relating to coal industry health benefits), or ``(2) 7.5 percent of the aggregate gross revenue from the sale of qualified metallurgical coal sold by the taxpayer during the taxable year to an unrelated person. ``(b) Qualification.--No credit shall be determined under subsection (a) with respect to any taxpayer unless at least 20 percent of the total tons of qualified coal sold by the taxpayer during each of the 4 preceding taxable years was qualified metallurgical coal. For purposes of the preceding sentence, only coal produced from an economic interest held by the taxpayer shall be taken into account. ``(c) Definitions and Special Rules.--For purposes of this section-- ``(1) Applicable percentage.--The applicable percentage is the percentage of the aggregate tons of qualified coal sold by the taxpayer during the taxable year to unrelated persons which consists of qualified metallurgical coal. In no event shall the applicable percentage exceed 75 percent. ``(2) Qualified metallurgical coal.--The term `qualified metallurgical coal' means any bituminous coal which-- ``(A) is used in the production of iron and steel, and ``(B) is processed by, or produced from an economic interest held by, a signatory operator. ``(3) Unrelated person.--The term `unrelated person' has the same meaning as when used in section 29. ``(4) Affiliated groups.--All members of the same affiliated group (as defined in section 1504) shall be treated as one taxpayer for purposes of this section and the credit determined under subsection (a) with respect to such one taxpayer shall be allocated among such members in such manner as the Secretary may prescribe. ``(5) Economic interest.--The term `economic interest' means an interest with respect to which an allowance for depletion is allowable. ``(6) Bituminous coal.--The term `bituminous coal' means coal classified as bituminous coal according to the publication of the American Society for Testing and Materials under the title `Standard Classification of Coals by Rank' (ASTM D 338- 91A), as in effect on the date of the enactment of chapter 99. ``(7) Qualified coal.--The term `qualified coal' means any bituminous coal which is produced and processed by a signatory operator from an economic interest held by a signatory operator. ``(8) Signatory operator.--The term `signatory operator' has the meaning given such term by section 9701(c)(1), and shall include all members of the affiliated group of which such signatory operator is a member that are also signatory operators. ``(9) Ton--.The term `ton' means 2,000 pounds.''. (b) Credit Made Part of General Business Credit.--Subsection (b) of section 38 is amended by striking ``plus'' at the end of paragraph (7), by striking the period at the end of paragraph (8) and inserting ``, plus'', and by adding at the end thereof the following new paragraph: ``(9) the metallurgical coal credit determined under section 45A.''. (c) Credit May Offset 90 Percent of Minimum Tax.--Subsection (c) of section 38 of such Code is amended by redesignating paragraph (2) as paragraph (3) and by inserting after paragraph (1) the following new paragraph: ``(2) Metallurgical coal credit may offset 90 percent of minimum tax.-- ``(A) In general.--In the case of a taxpayer entitled to a credit determined under section 45A, the amount determined under paragraph (1)(A) shall be reduced by the lesser of-- ``(i) the portion of the metallurgical coal credit not used against the normal limitation, or ``(ii) 90 percent of the taxpayer's tentative minimum tax for the taxable year. ``(B) Portion of metallurgical coal credit not used against normal limitation.--For purposes of subparagraph (A), the portion of the metallurgical coal credit not used against the normal limitation is the excess (if any) of-- ``(i) the portion of the credit under subsection (a) which is attributable to the metallurgical coal credit determined under section 45A, over ``(ii) the limitation of paragraph (1) (determined without regard to this paragraph) reduced by the portion of the credit under subsection (a) which is not so attributable.'' (d) Clerical Amendment.--The table of sections for subpart D of part IV of subchapter A of chapter 1 of such Code is amended by adding at the end thereof the following new item: ``Sec. 45A. Metallurgical coal mining credit.''. (e) Effective Date.--The amendments made by this section shall take effect on January 1, 1993. | Title: A bill to amend the Internal Revenue Code of 1986 to provide a tax credit to businesses which mine metallurgical coal and are required to make contributions to the UMWA Combined Benefit Fund created by the Energy Policy Act of 1992 Summary: Amends the Internal Revenue Code to allow a general business credit for metallurgical coal mining. Declares such credit to consist of: (1) the lesser of a percentage of coal industry health benefit premiums; or (2) a percentage of the sale of metallurgical coal. Allows such credit to offset a percentage of the minimum tax. | 1,327 | 130 | billsum | en |
Summarize: FIELD The present invention relates to a personal care product including a personal care composition, for example an antiperspirant composition or a deodorant composition, in the form of a solid composition, typically a stick. The present invention also relates to the manufacture of such a personal care product. BACKGROUND There are various techniques to provide a unique appearance to a packaged product. Many techniques are directed to the use of colored containers and attractive labeling. Another technique is to use the product to additionally provide part of the overall unique appearance of the product. It is known to provide personal care compositions having a random or non-random patterned appearance. Some of these compositions are an antiperspirant composition and/or a deodorant composition in the form of a stick. Nevertheless, there is a need in the art for a personal care composition, for example an antiperspirant composition and/or a deodorant composition, which has a distinctive aesthetic appearance as compared to known products. There is also a need in the art for a package for a personal care composition, for example an antiperspirant composition and/or a deodorant composition, that has a random diffuse pattern yet can be reliably and repeatably manufactured in large commercial volumes. There is furthermore a need in the art for a product comprising an antiperspirant composition and/or a deodorant composition, which is in stick form and exhibits improved aesthetic appeal. BRIEF SUMMARY An aim of this invention is to provide a package for a personal care composition, for example an antiperspirant composition or a deodorant composition, which can at least partially meet at least one of the needs identified above. Another aim of this invention is to provide a method for producing such a personal care composition, for example an antiperspirant composition or a deodorant composition, which can at least partially meet at least one of the needs identified above. The present invention accordingly provides personal care product, the product comprising a container containing a solid body of a topical personal care composition, the personal care composition having at least two components having differing visual characteristics, the components being mixed to form a random diffuse pattern, first and second components of the at least two components comprising major and minor proportions by volume of the solid body, the first component having a lower melting temperature than the melting temperature of the second component. The melting temperature of the first component may optionally be at least 23° C. lower than the melting temperature of the second component, for example 23 to 28° C. lower than the melting temperature of the second component. Typically, the personal care composition is an antiperspirant composition and/or a deodorant composition. The personal care composition may be a solid stick or soft solid. The personal care composition may comprise an antiperspirant active that is present in an amount of 5 to 25% by weight of the composition. Optionally, each of the at least two components of the personal care composition comprises the antiperspirant active. The personal care composition may comprise a deodorant active that is present in an amount of greater than 0 to up to 1% by weight of the composition. Optionally, each of the at least two components of the personal care composition comprises the deodorant active. The first and second components may respectively include different first and second gellant compositions to provide different respective melting temperatures of the first and second components of the personal care composition. Optionally, the first gellant composition comprises at least one soybean oil having an iodine value of greater than 0 to 20 and at least one fatty alcohol and the second gellant composition comprises at least one soybean oil having an iodine value of greater than 0 to 20 and at least one hydrocarbon of the formula C n H 2n+2, wherein n is 20 to 100, and the hydrocarbon is at least 90% linear. Typically, the iodine value is 1 to 5. The soybean oil may be present in an amount up to about 20% by weight of the respective component of the at least two components, typically in an amount up to 10% by weight of the respective component of the at least two components, more typically in an amount of 3 to 7% by weight of the respective component of the at least two components. The at least one fatty alcohol may be present in the first component in an amount of 5 to 25% by weight of the first component. Typically, the fatty alcohol comprises stearyl alcohol. The at least one hydrocarbon may be present in the second component in an amount of 5 to 25% by weight of the second component. Typically, the at least one hydrocarbon comprises polyethylene. Optionally, the personal care composition further comprises silicone which is present in each of the at least two components in an amount of 5 to 70% by weight of the respective component. Typically, the silicone comprises cyclomethicone. Optionally, the personal care composition further comprises an emollient chosen from PPG-14 butyl ether, C12-15 alkyl benzoate, phenyl trimethicone, PPG-3 myristyl ether, myristyl myristate, and combinations thereof. Typically, the at least two components have different colors. The present invention further provides a method of forming in a container a random diffuse pattern mixture of at least two components of a solid body of a topical personal care composition, the at least two components having differing visual characteristics, the method comprising the steps of: (a) providing a container on a container support capable of rotating the container; (b) feeding a first component of the at least two components into the container, the first component being molten and at a first temperature above a melting temperature of the first component; (c) at least after commencement of step (b), feeding a second component of the at least two components into the container, the second component being molten and at a second temperature above a melting temperature of the second component, the second temperature being higher than the first temperature and the melting temperature of the second component being higher than the melting temperature of the first component; (d) concurrently rotating the container on the container support in at least a first rotational direction during at least a part of feeding step (c) to form a random diffuse pattern mixture of the first and second components; and (e) cooling the first and second components to form a solid body having the random diffuse pattern mixture, the second component commencing solidification thereof during the cooling step prior to solidification of the first component. Optionally, the melting temperature of the first component is at least 47° C., typically 47 to 53° C., lower than the melting temperature of the second component. Optionally, the second temperature is at least 70° C., typically 70 to 76° C., higher than the first temperature. Typically, the first temperature is 47 to 53° C. and the second temperature is 70 to 76° C. For example, the first temperature may be from 62 to 69° C. and the second temperature may be from 79 to 86° C. Optionally, the second component terminates solidification thereof during the cooling step prior to solidification of the first component. Optionally, the first and second components respectively include different first and second gellant compositions to provide different respective melting temperatures of the first and second components of the personal care composition. Typically, the feeding of the second component in step (c) is commenced after termination of the feeding of the first component in step (b). Optionally, the container is rotated on the container support additionally during at least a part of feeding step (c). Optionally, the container is rotated on the container support after the termination of both feeding step (b) and feeding step (c). Optionally, the container is rotated on the container support additionally during at least a part of cooling step (e). The container is typically rotated on the container support in an oscillating manner in the first rotational direction and in an opposite second rotational direction. For example, the container is rotated at least 90 degrees in a first direction and at least 90 degrees in a second direction. Typically, the container is rotated up to about 360 degrees in a first direction and up to about 360 degrees in a second direction. The container may be rotated up to about 270 degrees in the first direction and up to about 270 degrees in the second direction. Optionally, the container support maintains the container at an angle of up to about 20 degrees to a vertical orientation. Optionally, the container is subject to a vibration during the feeding of at least the second component to the container. The first and second components may be fed into the container from a nozzle having two inputs and two outputs, one input and output pair for each respective component. In one aspect, the present processes can produce random diffuse patterns of personal care composition in containers. By random diffuse pattern is meant an irregular pattern that has a discernable artistic yet non-geometric pattern, but where the pattern varies in dimensions and the color varies in color density to provide a color gradation throughout the personal care composition. In one embodiment there may be strongly differentiated regions and/or lines of different color, for example a darker color composition dispersed randomly and diffusely in lighter color matrix. The results are unique and very artistic patterns. The solid personal care composition in the container may have a marbleized appearance or a swirl pattern. When the container is transparent or translucent, the pattern is continuously visible. When the container is opaque, the pattern is visible when the product is used, for example when a working surface of the topical personal care composition is revealed to the user, typically when the upper surface of a stick is exposed for topical application to the skin. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 schematically illustrates a front view of a personal care product including a personal care composition having a random diffuse pattern in accordance with an embodiment of the present invention. FIG. 2 is a schematic elevation view of a layout diagram of a process of filling a container according to a method of another embodiment of the present invention. FIG. 3 is a schematic elevation view of a layout diagram of a process of filling a container according to a method of a further embodiment of the present invention in which the support for the container is tilted at an angle to the filling nozzle. DETAILED DESCRIPTION It should be understood that the detailed description and specific examples, while indicating embodiments of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The citation of references herein does not constitute an admission that those references are prior art or have any relevance to the patentability of the invention disclosed herein. Any discussion of the content of references cited in the Background is intended merely to provide a general summary of assertions made by the authors of the references, and does not constitute an admission as to the accuracy of the content of such references. Each and every reference cited herein is hereby incorporated by reference in its entirety. The detailed description, while indicating embodiments of the invention, is intended for purposes of illustration only and is not intended to limit the scope of the invention. Moreover, recitation of multiple embodiments having stated features is not intended to exclude other embodiments having additional features, or other embodiments incorporating different combinations the stated of features. As used herein, the words “preferred” and “preferably” refer to embodiments of the invention that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful, and is not intended to exclude other embodiments from the scope of the invention. As used herein, the word “include”, and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the dispensers of this invention. The invention will now be described in more detail in its preferred embodiments with reference to the drawings. The described products and processes may be modified in minor details without departing from the concept of the present invention. As used throughout this description, ranges are used as a shorthand for describing each and every value that is within the range. Any value within the range can be selected as the terminus of the range. FIG. 1 schematically illustrates a front view of a personal care product including a personal care composition having a random diffuse pattern in accordance with an embodiment of the present invention. The personal care product, designated generally as 102, comprises a container 104 containing a solid body 106 of a topical personal care composition. The personal care composition is an antiperspirant composition and/or a deodorant composition, and may comprise an antiperspirant active that is present in an amount of 10 to 25% by weight of the composition and/or a deodorant active which is present in an amount of greater than 0 to up to 1% by weight of the composition. The container 104 comprises a conventional housing 108 having a cavity 110 for containing a solid body 106 formed as a hard stick or soft solid. A mechanism 112 for advancing or retracting the solid body 106 relative to the housing 108 is provided at the base 114 of the housing 108. A removable cap 116 may be fitted to the upper end 118 of the housing 108 to cover the exposed end 120 of the solid body 106. In FIG. 1, the solid body 106 has been advanced partly from out of the cavity 10 to illustrate the visual appearance of the solid body 106. The personal care composition has at least two components 122, 124, called first and second components, having differing visual characteristics. In alternative embodiments, greater than two visually distinct components may be provided. The at least two components may each comprise the antiperspirant active and/or the deodorant active. The two components differ in color. The first and second components 122, 124 are mixed to form a random diffuse pattern. The first and second components 122, 124 comprise major and minor proportions by volume of the solid body 106. Typically, the first component 122 comprises 70 to 95 wt %, typically 85 wt % and the second component 124 comprises 5 to 30 wt %, typically 15 wt %, of the solid body 106. Most typically, the second component 124 comprises the darker color composition dispersed randomly and diffusely in the lighter color matrix comprised of the first component 122. The first component 122 has a lower melting temperature than the melting temperature of the second component 124. Typically, the melting temperature of the first component 122 is at least 28° C. lower than the melting temperature of the second component 124. For example, the melting temperature of the first component 122 is 47 to 53° C. lower than the melting temperature of the second component 124. Having the components melt at different temperatures makes it easier to form striations or marbleized patterns in the composition. The higher melting point material solidifies first to start forming a solid that is mixed within the liquid lower melting point component. In certain embodiments, an inline mixer is not needed to mix the components. In this embodiment, the different melting temperatures of the first and second components 122, 124 is achieved by providing that the first and second components 122, 124 respectively include different first and second gellant compositions. For example, the first gellant composition comprises at least one soybean oil having an iodine value of greater than 0 to 20 and at least one fatty alcohol and the second gellant composition comprises at least one soybean oil having an iodine value of greater than 0 to 20 and at least one hydrocarbon of the formula C n H 2n+2, wherein n is 20 to 100, and the hydrocarbon is at least 90% linear. The soybean oil in the first and second components 122, 124 may be the same soybean oil. Typically, the iodine value is 1 to 5. The soybean oil may be present in an amount up to about 20% by weight, more typically up to about 10% by weight, for example 3 to 7% by weight, of the respective component of the at least two components. The at least one fatty alcohol may be present in the first component in an amount of 5 to 25% by weight of the first component. The fatty alcohol typically comprises stearyl alcohol. The at least one hydrocarbon may be present in the second component in an amount of 5 to 25% by weight of the second component. The at least one hydrocarbon may comprise polyethylene. The personal care composition typically further comprises silicone which is present in each of the at least two components in an amount of 5 to 70% by weight of the respective component. The silicone may comprise cyclomethicone. The personal care composition may further comprise an emollient chosen from PPG-14 butyl ether, C12-15 alkyl benzoate, phenyl trimethicone, PPG-3 myristyl ether, myristyl myristate, and combinations thereof. The composition is a solid stick or soft solid when at ambient room temperature of about 25° C. The stick form is an example of a solid form, and the soft solid is a thickened form that may or may not be solid. The stick form can be distinguished from a soft solid in that, in a stick, the formulated product can retain its shape for extended time periods outside the package, the product not losing its shape significantly (allowing for some shrinkage due to solvent evaporation). Adjustment of amounts of gelling or thickening agents can be used in order to form a soft solid or stick. Soft solids can be suitably packaged in containers that have the appearance of a stick, but which dispense through apertures (for example, slots or pores) on the top surface of the package. The soft solid products have also been called soft sticks or “smooth-ons”, and hereinafter are generically called “soft solids”. Reference is made to U.S. Pat. Nos. 5,102,656, 5,069,897, and 4,937,069, each of which discloses such soft solids, including physical characteristics thereof such as viscosity and hardness. The contents of each of these three U.S. patents are incorporated herein by reference to the extent that they do not conflict with the disclosure herein. Gelling Agents Gelling agents used in the topical personal care compositions used in the present invention comprise hydrogenated soybean oil as a first gellant and a second gellant comprising a fatty alcohol for the first component 122 or a hydrocarbon of the formula C n H 2n+2, wherein n is 20 to 100, and the hydrocarbon is at least 90% linear for the second component 124. The hydrogenated soybean oil is used as a co-gellant along with the selected second gellant to provide a solid stick or soft solid antiperspirant. The hydrogenated soybean oil is almost, but not fully hydrogenated. The amount of hydrogenation is measured by the iodine value. The iodine value can be measured by ASTM D5554-95 (2006). The iodine value of the hydrogenated soybean oil used herein is greater than 0 to 20. In one embodiment, the iodine value is 1 to 5. It has been found that this level of hydrogenation provides the desired structure to the antiperspirant and provides a softer and creamier application aesthetics. The hydrogenated soybean oil is present in an amount up to about 20% by weight of the composition. In another embodiment, the amount is up to about 10% by weight. In one embodiment, the amount is 3 to 7% by weight. In another embodiment, the amount is 4 to 6% by weight. The hydrogenated soybean oil can provide increased fragrance longevity when used to replace hydrogenated castor oil. The fatty alcohol can be any fatty alcohol. In one embodiment, the fatty alcohol is stearyl alcohol. The hydrocarbon is a hydrocarbon of the formula C n H 2n+2, wherein n is 20-100, and the hydrocarbon is at least 90% linear. In one embodiment, the hydrocarbon is a paraffin. In another embodiment, the hydrocarbon is polyethylene. An example of a polyethylene can be found in U.S. Pat. No. 6,503,491, which is incorporated herein by reference only for its disclosure of the polyethylene. In another embodiment, the polyethylene has a weight average molecular weight in of 300 to 3000 and a melting point of 50 to 129° C. In one embodiment, the second gellant, comprising the fatty alcohol or the hydrocarbon as discussed above, is present in the composition in an amount of 5 to 25% by weight of the composition. In another embodiment, the amount is 10 to 20% by weight. The formulations of the invention may further comprise additional gelling agents, which include, but are not limited to, waxes, esters of fatty acid and fatty alcohol, triglycerides, or other cosmetically acceptable materials, which are solid or semi solid at room temperature and provide a consistency suitable for application to the skin. When using the hydrogenated soybean oil as a co-gellant with the fatty alcohol gellant, the composition has increased fragrance retention as compared to known compositions containing hydrogenated castor oil. Volatile Silicone Compositions according to the present invention may include a volatile silicone. In one embodiment, the volatile silicone is a volatile cyclic polydimethylsiloxane (cyclomethicone), e.g., cyclopentasiloxane. By volatile material it is meant that the material has a measurable vapor pressure at ambient temperature. Preferably, the volatile cyclic polydimethylsiloxane is cyclomethicone. Various types of cyclomethicones may be used. Illustratively, and not by way of limitation, the volatile silicones are one or more members selected from cyclic polydimethylsiloxanes such as those represented by Formula I: where n is an integer with a value of 3-7, particularly 5-6. Illustrative examples of suitable cyclomethicones are DC-345 and DC-245, manufactured by Dow Corning Corporation, Midland, Mich. These types include a tetramer (octylmethylcyclotetrasiloxane) and a pentamer (decamethylcyclopentasiloxane). In one embodiment, the amount of volatile silicone in the composition is 5 to 70% by weight of the composition. In another embodiment, the amount is 25 to 45% by weight. Antiperspirant Active Materials When the composition includes an antiperspirant active, any of the known antiperspirant active materials can be utilized in the composition. Antiperspirant actives include, but are not limited to, aluminum chlorhydrate, aluminum chloride, aluminum sesquichlorohydrate, aluminum-zirconium hydroxychlorides, complexes or adducts of the above-mentioned active ingredients with glycol, such as propylene glycol (for example, “Rehydrol” II from Reheis Chemical Co.), and combinations thereof. Known aluminum-zirconium salts in combination with neutral amino acids, such as glycine (e.g., aluminum-zirconium tetrachlorohydrex Gly) can also be used. Generally, any of the Category I active antiperspirant ingredients, listed in the Food and Drug Administration's Monograph on Antiperspirant Drug Products for overall-the-counter human use (Oct. 10, 1973) can be used. In other embodiments, the antiperspirant active is an aluminum salt and/or an aluminum-zirconium salt, such as those described above, that are further stabilized by betaine and a calcium salt. More information about betaine and calcium salt stabilized antiperspirant salts can be found in U.S. Patent Application Publication No. 2006/0204463 to Tang et al., which is incorporated herein by reference only for the disclosure of the antiperspirant actives. In other embodiments, the antiperspirant active, such as those described above, is selected to have a low metal to chloride ratio. Examples of these antiperspirant actives can be found in U.S. Pat. No. 6,375,937 to Chopra et al. and in U.S. Patent Application Publication No. 2004/0109833 to Tang et al., which are incorporated herein by reference only for their disclosure of the antiperspirant active. In other embodiments, the type of salt of interest, an aluminum zirconium tetrasalt or octasalt free of glycine are used wherein aluminum zirconium salt is stabilized by Betaine and has a metal to chloride ratio of 0.9:1 to 1.3:1 (and in other embodiments of 0.9:1 to 1.2:1 or 0.9:1 to 1.1:1). For the tetrasalt, the Al/Zr atomic ratio can be 3.2:1 to 4.1:1.0 and the Betaine:zirconium mole ratio can be 0.2:1 to 3.0:1 (or in other embodiments of 0.4:1 to 1.5:1). Another salt that can be used is an aluminum chloride salt buffered by Betaine, wherein the salt has a metal to chloride ratio of 0.9:1 to 1.3:1 (and in other embodiments of 0.9:1 to 1.2:1 or 0.9:1 to 1.1:1). For the octasalt the Al/Zr atomic ratio is 6.2:1 to 10.0:1 and the Betaine:Zr mole ratio is 0.2:1 to 3.0:1 (or in other embodiments of 0.4:1 to 1.5:1). In one embodiment, in the case of a salt that contains zirconium, the Betaine is incorporated during the synthesis of the salt so as to maximize the stabilizing effect this ingredient has (especially on the zirconium species). Alternatively, it can be post added to a glycine-free salt along with additional active phase ingredients to form a Betaine stabilized active. Examples of commercially available glycine-free low M:Cl ratio tetrasalts and octasalts include, but are not limited to, REZAL™ AZP 955 CPG and REZAL™ AZP 885 respectively (both from Reheis Chemical Company, Berkeley Heights, N.J.). A more detailed description of making such commercially available salts can be found for example, in U.S. Pat. Nos. 7,074,394 and 6,960,338. Further examples of making these types of salt complexes are described in U.S. Patent Application Publication No. 2004/0198998 and U.S. Pat. No. 7,105,691. In addition to the anti-irritation properties of Betaine, it has also been found that antiperspirant formulations preserve their fragrance stability upon ageing when the Al/Zr salt is used in association with Betaine. Additionally, the antiperspirant active can be a calcium salt stabilized antiperspirant active. Examples of calcium salt stabilized antiperspirant actives can be found in U.S. Patent Application Publication No. 2006/0204463, which is incorporated herein by reference only for the disclosure of the calcium salt stabilized antiperspirant actives. In addition, aluminum nitratohydrate and its combination with zirconyl hydroxychlorides and nitrates, or aluminum-stannous chlorohydrates, can be incorporated as an antiperspirant active. Antiperspirant actives can include, but are not limited to, the following: astringent salt of aluminum, astringent salt of zirconium, aluminum bromohydrate, aluminum chlorohydrate, aluminum dichlorohydrate, aluminum sesquichlorohydrate, aluminum chlorohydrex PG, aluminum dichlorohydrex PG, aluminum sesquichlorohydrex PG, aluminum chlorohydrex PEG, aluminum dichlorohydrex PEG, aluminum sesquichlorohydrex PEG, aluminum chloride, aluminum sulfate, aluminum zirconium chlorohydrate, aluminum zirconium trichlorohydrate, aluminum zirconium tetrachlorohydrate, aluminum zirconium pentachlorohydrate, aluminum zirconium octachlorohydrate, aluminum zirconium tetrachlorhydrex propylene glycol, aluminum zirconium trichlorohydrex Gly, aluminum zirconium tetrachlorohydrex Gly, aluminum zirconium pentachlorohydrex Gly, aluminum zirconium octachlorohydrex Gly, buffered aluminum sulfate, potassium alum, sodium aluminum chlorohydroxy lactate. In one embodiment, the antiperspirant active is aluminum chlorhydrate. In another embodiment, the antiperspirant active is aluminum zirconium tetrachlorhydrex propylene glycol. Deodorant Active Materials Any known deodorant active can be used. Examples of deodorant active include, but are not limited to, antimicrobial actives, alcohols, 2,4,4′-trichloro-2′-hydroxy diphenyl ether (Triclosan), benzethonium chloride, polyhexamethylene biguanides, triethylcitrate, 2-amino-2-methyl-1-propanol (AMP), cetyl-trimethylammomium bromide, cetyl pyridinium chloride, farnesol (3,7,11-trimethyl-2,6,10-dodecatrien-1-ol), bactericides, and/or bacteriostats. Emollients The composition can contain emollients in any desired amount to achieve a desired emollient effect. Emollients are known in the art and are used to impart a soothing effect on the skin. Non-volatile emollients are preferable in the present invention. Classes of non-volatile emollients include non-silicone and silicone emollients. Non-volatile, non-silicone emollients include C 12-15 alkyl benzoate. The non-volatile silicone material can be a polyethersiloxane, polyalkyarylsiloxane or polyethersiloxane copolymer. An illustrative non-volatile silicone material in the present invention is phenyl trimethicone. Non-limiting examples of emollients can be found in U.S. Pat. No. 6,007,799. Examples include, but are not limited to, PPG-14 butyl ether, PPG-3 myristyl ether, stearyl alcohol, stearic acid, glyceryl monoricinoleate, isobutyl palmitate, glyceryl monostearate, isocetyl stearate, sulphated tallow, oleyl alcohol, propylene glycol, isopropyl laurate, mink oil, sorbitan stearate, cetyl alcohol, hydrogenated castor oil, stearyl stearate, hydrogenated soy glycerides, isopropyl isostearate, hexyl laurate, dimethyl brassylate, decyl oleate, diisopropyl adipate, n-dibutyl sebacate, diisopropyl sebacate, 2-ethyl hexyl palmitate, isononyl isononanoate, isodecyl isononanoate, isotridecyl isononanoate, 2-ethyl hexyl palmitate, 2-ethyl hexyl stearate, Di-(2-ethyl hexyl)adipate), Di-(2-ethyl hexyl)succinate, isopropyl myristate, isopropyl palmitate, isopropyl stearate, octacosanol, butyl stearate, glyceryl monostearate, polyethylene glycols, oleic acid, triethylene glycol, lanolin, castor oil, acetylated lanolin alcohols, acetylated lanolin, petrolatum, isopropyl ester of lanolin, fatty acids, mineral oils, butyl myristate, isostearic acid, palmitic acid, PEG-23 oleyl ether, olelyl oleate, isopropyl linoleate, cetyl lactate, lauryl lactate, myristyl lactate, quaternised hydroxy alkyl, aminogluconate, vegetable oils, isodecyl oleate, isostearyl neopentanoate, myristyl myristate, oleyl ethoxy myristate, diglycol stearate, ethylene glycol monostearate, myristyl stearate, isopropyl lanolate, paraffin waxes, glycyrrhizic acid, hydrocyethyl stearate amide. The composition can additionally include ionizable inorganic salts. These ionizable salts are of the form M a X b where a=1, or 2 and b=1 or 2; M is a member chosen from Na +1, Li +1, K +1, Mg +2, Ca +2, Sr +2, and Zn +2 and X is a member chosen chloride, bromide, iodide, citrate, gluconate, lactate, glycinate, glutamate, ascorbate, aspartate, nitrate, phosphate, hydrogenphosphate, dihydrogenphosphate, formate, maloneate, maleate, succinate, carbonate, bicarbonate, sulfate, and hydrogensulfate. In certain embodiments, the selected salts are chosen from NaCl and ZnCl 2. As will be appreciated by those skilled in the art, while it may be possible under certain circumstances to add a salt directly to a portion of the mixture during manufacturing, it is desired to add the salt as a mixture or solution of the salt in a carrier or solvent, particularly water. Of course various concentrations of the salt premix can be made. The composition may also contain particulates which include but are not limited to talc, mica, fragrance encapsulates, or hydrophobically modified starches, such as aluminum starch octenyl succinate (MACKADERM™ ASTRO-DRY™ from McIntyre Group Ltd.). If the composition is in a liquid form and dispensed through a roll-on applicator, the average particle size of the suspended material is sized so that it can pass through the application to prevent the ball applicator from malfunctioning. Usually, the average particle size does not exceed 150 microns. In certain embodiments, the composition may also contain as an optional ingredient at least one malodor counteracting alpha, beta-unsaturated ester or mixtures of such materials. In certain embodiments, the level of malodor counteracting composition to deliver a perceivable odor control benefit when delivered from an antiperspirant and/or deodorant composition is 0.05 to 0.45 weight % based on the entire composition. The alpha, beta-unsaturated ester malodor counteracting materials are incorporated within the oil phase of an antiperspirant composition. Example of these malodor counteracting components can be found in U.S. Pat. Nos. 6,610,648 and 6,495,097, which are incorporated herein only for their disclosure of the alpha, beta unsaturated esters. For example, in this invention the odor neutralizing alpha, beta unsaturated ester mixture demonstrates unexpected stability in antiperspirant compositions containing low metal:chloride (M:Cl) ratio salts free of glycine. Examples of the alpha, beta unsaturated ester can be found in WO2005/025523, which was filed in the U.S. as U.S. application Ser. No. 10/571,488, both of which are incorporated herein by reference to the extent that they do not conflict with the disclosure in this specification. Examples of the alpha, beta unsaturated ester include, but are not limited to: (1) 3-phenyl-2-propenoic acid alkyl esters wherein R 1 is a substituent on the benzene ring and is chosen from an alkyl, an alkoxy, an aryl, or a substituted aryl. In certain embodiments, R 1 is chosen from H, a C 1 to C 8 alkyl, a C 1 to C 8 alkoxy, or an aryl; and R 2 is a subsistent group replacing the carboxylic acid hydrogen to form the ester where R 2 has greater than 6 carbon atoms, an aryl, or a substituted aryl group, in certain embodiments R 2 is a C 6 to C 12 alkyl or is a benzyl group; and (2) an ester of fumaric or maleic acid having linear ester carbon chains from 3-9 carbons, for example dihexyl fumarate; (3) e-phenyl propenoic acid ester chosen from octyl methoxy cinnamate, phenylethyl cinnamate, benzyl cinnamate; (4) an aliphatic unsaturated ester, such as dihexyl fumarate. The composition may optionally further comprise absorbent materials such as corn starch, talc, clay, sodium polyacrylate and/or cotton fiber; and/or other materials such as fragrances, bacteriostats and/or bacteriosides, colorants, etc. Known bacteriostats include baceteriostatic quaternary ammonium compounds such as 2-amino-2-methyl-1-propanol (AMP), cetyl-trimethylammomium bromide, cetyl pyridinium chloride, 2,4,4N-trichloro-2N-hydroxydiphenylether (Triclosan), etc. and various zinc salts. Antioxidants may be added to the composition, preferably to act as ingredient protectants and for maintenance of long-term stability of the composition. Suitable antioxidants include Tinogard, manufactured by Ciba Specialty Chemicals, Basel, Switzerland. The compositions as provided herein are described and claimed with reference to their ingredients, as is usual in the art. As would be evident to one skilled in the art, ingredients may in some instances react with one another, so that the true composition of the final formulation may not correspond exactly to the ingredients listed. Thus, it should be understood that the invention extends to the product of the combination of the listed ingredients. The individual component compositions of the present invention may be manufactured using method steps generally known in the art. Typically, the ingredients are combined and heated to melt the components (other than inert filler), and the melted components (together with particulate inert filler) are mixed. Desirably, volatile materials, such as the fragrance materials, are incorporated in the composition in the latter stages of the mixing cycle, in order to avoid volatilization thereof. After mixing, the molten composition can be poured directly into the dispensers, as discussed herein in detail concerning the sequential pouring of the two components, after which the compositions harden into a solid, and the container is capped to preserve the product until use. Method for Filling the Personal Care Composition into a Container This invention also relates to a method for filling into a container a multi-component topical personal care composition in a diffuse pattern where the components have at least one visually discernable different characteristic, such as a marbleized appearance. This produces a solid body of the topical personal care composition which has an aesthetic appearance to the purchaser and/or user of the product. More particularly, the container, or a portion thereof such as a removable cap, may be transparent or translucent, such that the aesthetic topical personal care composition presents a unique appearance at the exterior of the container prior to use, or if the container is opaque, such that the aesthetic topical personal care composition presents a unique appearance at the exposed portion of the composition for application to the skin during use. The present process will produce containers filled with two or more components in a diffuse pattern design. The precise patterns and the intensity of the patterns are the result of the process parameters in the filling of the containers, and the properties of the components of the topical personal care composition. The process parameters include, inter alia, the temperatures, and associated melting temperature, of the first and the second components which are filled, as molten liquids, into the container, the amount of each of the first component and of the second component the shape of the container, and motion of the container during the filling process, for example the degree and rate of oscillation of the container. FIG. 2 is a layout diagram of one embodiment of the filling apparatus. In FIG. 2 a container 15 is at an early stage of being filled with the first component 30 in molten form. There are two separate components needed to produce the product in the container 15. These are a first component 10 and a second component 20. The first component 10 and the second component 20 are visually distinct from each other. The first component 10 is fed into a heated reservoir 16 through an input conduit 18. Heated reservoir 16 maintains the first component at a respective preset temperature in molten form. The first component exits the heated reservoir 16 through an exit conduit 14 to a valve 17. The first component 10 flows from the valve 17 though feed conduit 12 and then through a first component output conduit 19 of a nozzle 25 having an outlet 31. The valve 17 controls the time of injecting for the first component 10 and a flow meter (not shown) may measure the required volume corresponding to the desired mass of that component to be injected into the container 15. The second component 20 is correspondingly fed into a heated reservoir 26 through an input conduit 28. Heated reservoir 26 maintains the second component at a respective preset temperature in molten form. The second component exits the heated reservoir 26 through an exit conduit 24 to a valve 27. The second component 20 flows from the valve 27 though feed conduit 23 and then through a second component output conduit 29 of the common nozzle 25. The valve 27 controls the time of injecting for the second component 20 and a flow meter (not shown) may measure the required volume corresponding to the desired mass of that component to be injected into the container 15. All of the conduits are thermally insulated as required to ensure that the components 10, 20 are injected onto the container 15 in a molten state at the required temperatures. Initially, the required measured volume of the first component 10 is injected to partly fill the container 15. Subsequently, the required measured volume of the second component 20 is injected into the container 15 to completely fill the container 15. The first component 10 is at a lower temperature than the second component 20. As the composition cools, for example by application of forced cooling, such as by passage through a cooling tunnel, the second component 20 solidifies within the molten first component 10. During injection of at least the second component 20 and preferably also during injection too of the first component 10, the container 15 is supported on a rotatable support 13 that is rotated throughout the filling process, and optionally for a short period hereafter. While any degree of rotation can be used, the rotation is typically in a first direction through at least 90 degrees, and then in a second direction through at least 90 degrees. In order to achieve the desired marbleized random pattern, preferably the container 15 is first rotated in a first direction and then in a second direction in an oscillating motion. The oscillations of a rotation in a first direction and then in a second direction may be related to the flow rate of the first component 10 and second component 20 into the container 15 to fill the container 15. The container is typically rotated at least 90 degrees in the first direction and at least 90 degrees in the second direction, preferably at least about 180 degrees in the first direction and at least about 180 degrees in the second direction. The nozzle may extend within the container at the initiation of the filling of the container and be separated from the container during the filling of the container by relative withdrawal of the nozzle from the container or the container being withdrawn from the nozzle. During this process, the nozzle 25 is maintained above fill level 33 of the product 30 in the container 15. This may be accomplished by either raising the nozzle 25 upward or by lowering the container support 13. It is preferred to raise the nozzle 25. The oscillations may be through about 120 degrees to about 480 degrees and may comprise about 1 oscillation to about 10 oscillations and preferably about 2 to 7 oscillations to fill a container 15. FIG. 3 is a further embodiment of the filling apparatus of FIG. 2 but with the rotatable container support 13 tilted at an angle to the horizontal so that the container axis is inclined to the nozzle 25 and to the vertical by an angle of typically up to about 20 degrees. Thus the container 15 is tilted at an angle to the nozzle 25 as it is being rotated and filled. The angling may be at an angle of about 3 degrees to about 20 degrees, typically up to about 15 degrees, to the nozzle 25. In another embodiment, not illustrated, the support 13 includes a device to vibrate the support 13 and thereby vibrate the container 15. The vibration may occur while the container 15 is being rotated. The result is that the container 15 is being vibrated while the container 15 is being oscillated and filled with the first component and the second component to produce the random pattern composition 30. The vibration and oscillation do not have to occur at the same time. Additionally, it is not required that the container 15 be oscillated in this embodiment of the invention. The vibration of the support 13 and the container 15 during the filling of the container will cause the pattern of the product 30 in the container 15 to become more diffuse and will promote product 30 as it exits nozzle 25 to flow away from nozzle 25 to parts of the container that are more distant from the nozzle 25. This will be useful in filling non-circular containers such as oval containers that have an elliptical cross-section. It also will be useful in the filling of non-axial containers. These are containers that are not symmetrical around the axis of the container formed through the container fill and dispensing opening. Both the amplitude and the frequency of the vibrations will depend on the particular formulations. The color of the first component 10 and the second component 20 may vary. However, the objective usually will be to use contrasting colors so as to make the diffuse design more vibrant and visible. A useful pairing of two components is to have one white and the other a color. With color matching the variations are essentially unlimited. Further, there can be more than two components sequentially injected into the container. There can be three or more components, and in addition, particles or capsules may be included. This will provide a wider range of diffuse patterns to products. The container 15 may be of essentially any shape, size or material construction. Since the stick products, such as antiperspirant/deodorant stick products, will primarily be consumer product-sized, the containers will contain 25 ml to 2 liters, typically 50 to 100 ml, of product and may be constructed of polyethylene, clarified polypropylene, polyethylene terephthalate and polyvinyl chloride. EXAMPLE In the following is set forth an example of the present invention. The example is illustrative, and not limiting, of the present invention. In the following example, all amounts are in percent of the total weight of the composition. First and second antiperspirant components having the compositions in Table 1 are made using known formulation techniques. Component 1 uses stearyl alcohol as the fatty alcohol co-gellant with hydrogenated soybean oil to produce a relatively low melting point component having a melting point of 47-53° C. Component 2 uses polyethylene as the hydrocarbon co-gellant to produce a alcohol co-gellant with hydrogenated soybean oil to produce a relatively high melting point component having a melting point of 70-76° C. The iodine value of the hydrogenated soybean oil used in the examples is greater than 0 to 1. The weight average molecular weight of the polyethylene used in the examples is indicated by the product number. TABLE 1 Stick Composition Component 1 Component 2 Cyclomethicone (DC245 from Dow 15.2 13.6 Corning) C 12-15 alkyl benzoate 6 17.1 Stearyl alcohol 15.8 0 Polyethylene 0 11 PEG-8 distearate 3.4 4.3 Hydrogenated soybean oil 3.4 4.3 Antiperspirant Active Z576 22.4 Antiperspirant Active 908 20.4 0 PPG-14 butyl ether 6 0 Talc 0 0 Behenyl alcohol 0.2 0 Blue Cosmetic Wax (or FD&C Green) 0 0.2 Component 1 is heated to a temperature of 66° C., which is above the melting point of Component 1. Component 2 is heated to a temperature of 82° C., which is above the melting point of Component 2. Both components are stored in respective temperature controlled heated reservoirs having a rotary hydrofoil blade rotating at 60-70 rpm. An inverted barrel of a conventional stick container is positioned on a rotatable support plate that is oscillatingly rotated. A measured amount, corresponding to 85 wt % of the final stick product, of the molten Component 1 is injected downwardly into the inverted barrel. Then, after completion of the injection of Component 1, a measured amount, corresponding to 15 wt % of the final stick product, of the molten Component 2 is injected into the molten mass of Component 1. A base is applied to the barrel, and then the barrel is transferred to a cooling tunnel, set at 17-18° C., and allowed to cool for 15-20 minutes prior to conditioning at ambient temperature, and final product labeling. As the composition cools, Component 2 starts to solidify within the still molten Component 1 and completes solidification before solidification of the lower melting point Component 1. The result is a marbleized appearance throughout the composition. The applied forced cooling of the composition assists minimization of migration or bleeding of the higher melting temperature colored phase into the lower melting temperature white background phase. Various other modifications to the disclosed embodiments will be apparent to those skilled in the art. In particular, alternative embodiments having a cap fitted to a container so that the package may be selectively stood upright either on its container end or on its cap end may comprise stick dispensers, for dispensing a solid stick of a personal care composition such as an antiperspirant composition and/or a deodorant composition, rather than roll-ball dispensers. | Summary: Disclosed is a package for a personal care composition, the package comprising a container containing a personal care composition, the container having a base comprising a base face for standing the package on a planar surface, and a cap removably connected to and covering a dispensing end of the container opposite to the base. The cap has an end face which is inclined to the base face, and the container and cap are shaped to permit the package, independent of the amount of the personal care composition contained within the container, selectively to be stood on the base face in an upright orientation on the planar surface or on the end face in an inverted orientation on the planar surface. | 11,688 | 143 | big_patent | en |
Summarize: By. Chris Pleasance. PUBLISHED:. 16:57 EST, 3 December 2013. |. UPDATED:. 08:36 EST, 4 December 2013. Another large oil spill has hit Nigeria's already devastated Niger Delta region, killing precious mangrove swaps and wildlife. While the spill happened near ENI's Brass facility, the company says it has been unable to confirm the cause of this latest disaster. Many spills in the region are caused by pipe sabotage or theft from the pipelines, as many people live in poverty and try to cash in on the valuable natural resource. A large oil spill near ENI's Brass facility on the Niger Delta was reported on Monday, similar to this one which was caused by oil thieves in March. Oil spills are a recurring problem in the area, with precious mangrove swamps, home to some of the planet's rarest wildlife, being left blackened and dead as a result. While many spillages are the result of theft from the pipelines by a population which lives mostly in poverty, many of them are due to loading accidents and decrepit infrastructure. The problem has become so bad that Nigeria's Government is considering tough new laws to fine companies that are found to be responsible for the disasters. If passed the legislation could cause Western-owned companies to be fined hundreds of millions of dollars each year. However loading accidents, coupled with decrepit infrastructure, have also been found to be behind several, with companies usually forced to pay for a cleanup operation and for compensation to the locals affected. The Government is now considering passing laws which would impose harsher fines on those responsible, meaning many Western-owned businesses could incur fines totalling tens of millions of dollars every year. Vast stretches of the delta's unique mangrove swamps, home to some of the planet's rarest wildlife, are blackened and dead from oil pollution. At the moment companies found to be responsible are forced to pay for a cleanup operation and for compensation for any locals affected. Oil is the largest contributor to the economy in Africa's most populous nation but has been marred by corruption since the reserves were discovered in the 1950s. Those who live around the Delta rely on fishing for their main source of income, an industry badly damaged by the spills which wipe out life in the rivers. Royal Dutch Shell is the biggest operator in the region, followed by ENI. In 2000 their trade accounted for 98 per cent of all export revenue in Nigeria. According to the Huffington Post, ENI said in a statement: 'During loading operations on a tanker on Nov. 27, an oil spill in the sea was seen. Operations were immediately suspended and resumed only after it was verified that the vessel's structures were not damaged and were not leaking.' Francis Clinton Tubo Ikagi, chairman of the Odioama fishing community in Bayelsa, where a large part of the Niger river fans out through creeks into the Atlantic, told journalists on the scene that he saw a large oil slick on Nov. 20. 'I saw a very thick layer of crude oil on the river,' he said. 'The community is affected seriously. Our women and men whose main livelihood source is fishing are complaining bitterly to us that the whole river is full of oil.' Hi-lighting the extent of the theft, this illegal refinery was destroyed by the Joint Task Force at Nembe Creek in Niger Delta in March. In 2011, the Nigerian government said there had been more than 7,000 oil spills in the country between 1970 and 2000 that could take up to 30 years and $1billion to clean up. Fishing boats were abandoned on Bodo waterways which were polluted by spilled crude oil allegedly caused by Shell equipment failure in Ogoniland in 2010. Corruption among Nigeria's politicians has meant that only a small amount of the profits made from oil have trickled down into the communities from which it is taken. Estimates of Nigeria's oil reserves make it around the tenth most oil-rich nation on Earth and the most affluent in Africa. In 2011, the Nigerian government said there had been more than 7,000 oil spills in the country between 1970 and 2000 that could take up to 30 years and $1billion to clean up. In Nigeria's Akwa Ibom State, an ExxonMobil pipeline ruptured on May 1 2010 and spilled over a million gallons of oil. The leak continued for seven days before it was stopped. In December 2011 there was another spill near the coast of Nigeria, and was was reported as 'likely the worst to hit those waters in a decade.' Here fluorescent trickles of oil can be seen in the main river while to the right, more devastation can be seen in the blackened forest. Shell was the first company to strike oil in Nigeria at Oloibiri and in 1958 began drilling. This is Shell's Awobo flow station in the Niger Delta. Nigeria has the highest concentration of mangrove forests in Africa, covering nearly 14,000sq miles, though many have been destroyed by the spills. Many communities, such as this one, are surrounded by rivers and are almost wholly dependent on them for survival, so are badly affected when oil spills occur. After two days, it had affected 115 miles (185 kilometers) of Nigerian coastline. Several days after the December 20 spill, Shell reported that the leak -- which occurred about 75 miles offshore -- had been contained before it reached the Nigerian coast. The spill, which covered 350 square miles of ocean at its peak, was reported as having released less than '40,000 barrels -- or 1.68 million gallons' of oil | Summary: Another large oil spill was reported near an ENI facility in Nigeria yesterday. The company has said it cannot tell what was behind the latest incident. The Government is now considering harsher fines for companies behind the disasters. Local communities which rely heavily on fishing have been badly affected. Theft from pipelines as well as loading accidents and decrepit infrastructure is to blame. Nigeria is Africa's most affluent nation and the tenth most oil rich in the world. | 1,349 | 99 | cnn_dailymail | en |
Write a title and summarize: Plague is still a public health problem in the world and is re-emerging, but no efficient vaccine is available. We previously reported that oral inoculation of a live attenuated Yersinia pseudotuberculosis, the recent ancestor of Yersinia pestis, provided protection against bubonic plague. However, the strain poorly protected against pneumonic plague, the most deadly and contagious form of the disease, and was not genetically defined. The sequenced Y. pseudotuberculosis IP32953 has been irreversibly attenuated by deletion of genes encoding three essential virulence factors. An encapsulated Y. pseudotuberculosis was generated by cloning the Y. pestis F1-encoding caf operon and expressing it in the attenuated strain. The new V674pF1 strain produced the F1 capsule in vitro and in vivo. Oral inoculation of V674pF1 allowed the colonization of the gut without lesions to Peyer' s patches and the spleen. Vaccination induced both humoral and cellular components of immunity, at the systemic (IgG and Th1 cells) and the mucosal levels (IgA and Th17 cells). A single oral dose conferred 100% protection against a lethal pneumonic plague challenge (33×LD50 of the fully virulent Y. pestis CO92 strain) and 94% against a high challenge dose (3,300×LD50). Both F1 and other Yersinia antigens were recognized and V674pF1 efficiently protected against a F1-negative Y. pestis. The encapsulated Y. pseudotuberculosis V674pF1 is an efficient live oral vaccine against pneumonic plague, and could be developed for mass vaccination in tropical endemic areas to control pneumonic plague transmission and mortality. Plague, the dreadful infectious disease that caused three major pandemics in history, is still a public health problem. Since the 1980s, an increase of cases worldwide has been observed, leading to categorize plague as a re-emerging disease. Whereas the most active foci of human plague are located in east-central Africa and Madagascar [1], recent cases have also been recorded in areas from where it had disappeared for decades, like Algeria, Libya, Zambia and Jordan [2], [3], [4]. Moreover, because plague is principally a zoonotic disease affecting rodents, the territories where it is endemic in its animal reservoir are much more extended than the observed human plague foci. Plague is an acute, often fatal infection whose etiologic agent, Yersinia pestis, is a Gram-negative bacillus with an extreme pathogenicity. The accidental transmission of the plague bacillus from rodents to humans by the bite of infected fleas is the most frequent mode of human infection. From the skin, the bacteria migrate to the draining lymph nodes, causing the bubonic form of plague, which evolves toward septicemia and death within one week if treatment is not rapidly started. When the bacteria reach the lungs, an acute pneumonia develops. Patients become highly contagious through the emission of infected aerosols that cause primary pneumonic plague. The disease is then generally fatal within 3 days or less. Human-to-human transmission of pneumonic plague favors a rapid spread of the disease in heavily populated areas, as occurred for example recently in Madagascar and in the Democratic Republic of Congo [5], [6]. Although antibiotherapy is currently the main tool to fight the disease, the residual plague mortality in endemic countries remains around 10% or more. This is mainly due to the difficulty for patients from areas far from health structures to receive the treatment on time. An additional cause of concern is the recent identification in Madagascar of two Y. pestis strains naturally resistant to antibiotics, one of which was resistant to eight different antibiotics, including those recommended for plague treatment and prophylaxis [7]. Because this multi-drug resistant Y. pestis resulted from the acquisition of a widespread self-transmissible plasmid [8], the rise of such threatening Y. pestis variants may be anticipated. Finally, Y. pestis is also classified in the list A of pathogens with potential for bioterrorist use established by the US Center for Disease Control due to its pathogenicity and human-to-human transmission [9], and the possibility that the bacteria is engineered to resist to antibiotics for evil use cannot be excluded. In front of such a public health risk, mass vaccination might be one of the only alternatives to protect exposed populations. However, no safe and efficient vaccine against plague is currently available. The first widely used plague vaccine was the live attenuated Y. pestis EV76 developed in Madagascar. This vaccine, that can have severe secondary effects, is now used in only few countries such as China or the former USSR. The licensed killed whole-cell Y. pestis vaccine from Greer/Miles was recently discontinued because it was reactogenic in humans and conferred only short-term protection [10], requiring annual booster immunizations. Much effort has been made in the recent years to develop new candidate vaccines. The strategies followed to induce protective immunity include the attenuation of live Y. pestis by genetic engineering, the introduction of Y. pestis antigens in Salmonella [11] and virus vectors [12], [13], as well as the production of subunit vaccines combining the capsular F1 antigen and the V antigen (LcrV) [14], [15], [16]. We recently reported that the attenuated Y. pseudotuberculosis strain IP32680 can be used as a live oral vaccine against bubonic plague [17]. The rationale for choosing this approach was to combine the immunogenicity and antigenic complexity of live vaccines with the much lower virulence of Y. pseudotuberculosis. Indeed, Y. pestis is a clone recently emerged from Y. pseudotuberculosis [18], and the two species share more than 95% genetic identity. A reason not to use Y. pestis as live vaccine is its genetic instability, as revealed by the spontaneous genome reductions observed for the EV76 strain, which hampered its vaccine efficiency [19]. That risk is much lower for Y. pseudotuberculosis because such rearrangements in Y. pestis are thought to result from the high number of insertion sequences (IS) present in its genome [20], and Y. pseudotuberculosis has a much lower number of IS copies and so is genetically much more stable [20]. When given orally, Y. pseudotuberculosis IP32680 was able to colonize the gut without causing lesions and stimulated a protective immune response against bubonic plague [17]. These results demonstrate the feasibility of using a live attenuated Y. pseudotuberculosis strain as an oral vaccine against plague. However, IP32680 is not suitable for human use because the genetic bases of its attenuation are not known and it does not confer high-level protection against pneumonic plague. The aim of the present study was to construct a genetically engineered Y. pseudotuberculosis strain irreversibly attenuated in virulence, and able to confer high-level protection against pneumonic plague. Animals were housed in the Institut Pasteur animal facilities accredited by the French Ministry of Agriculture to perform experiments on live mice (accreditation B 75 15-01, issued on may 22nd, 2008), in appliance of the French and European regulations on care and protection of the Laboratory Animals (EC Directive 86/609, French Law 2001-486 issued on June 6,2001). Protocols were approved by the veterinary staff of the Institut Pasteur animal facility and were performed in compliance with the NIH Animal Welfare Insurance #A5476-01 issued on 02/07/2007. The Y. pseudotuberculosis and Y. pestis isolates used in this study and their derivatives are listed in Table 1. Bacteria were grown at 28°C on Luria-Bertani agar plates supplemented with 0. 2% hemin (LBH) for 48 h before use, and bacterial concentrations were evaluated by spectrometry at 600 nm and plating on LBH plates. Deletion of the caf operon was performed in Y. pestis CO92p (Table 1) using the Short Flanking Homology (SFH) procedure [21] with primers 812 and 814 designed to exchange a portion of the caf locus encompassing the caf1M, caf1A and caf1 genes by a kanamycin resistance cassette (km from plasmid pGP704N-km). Deletion of the Y. pseudotuberculosis High Pathogenicity Island (HPI: YPTB1585 through YPTB1602), yopK (virulence plasmid gene PYV0040) and psaA (YPTB1334) sequences from IP32953p was done by allelic exchange with a kanamycin (km from plasmid pUC4K), spectinomycin (spec from plasmid pSW25) and trimethoprim (dfr from plasmid pGP704N-dfr) resistance cassette, respectively. HPI deletion was done following the Long Flanking Homology procedure [21]. yopK and psaA genes were first deleted individually in Y. pestis CO92p (Table 1) following the SFH procedure [21]. Second, the genomic DNA from the resulting deletants was used as template for PCR amplification of the antibiotic resistance cassettes flanked by the 500 bp upstream and downstream regions of yopK and psaA genes. The PCR products were electroporated into Y. pseudotuberculosis IP32953ΔHPI (Table 1), as described previously [21]. Recombinant colonies were selected for antibiotic resistance and were verified by PCR with primers located: (i) on each side of the inserted antibiotic cassette, and (ii) within each target region (Table S1). All the primer pairs used to generate PCR products for allelic exchange are listed in Table S1. To clone the caf operon into pGEN-lux [22], the entire locus was amplified with primer pair 837/838, which adds NotI and ApaI sites at the extremities. The PCR product was ligated to the corresponding sites in place of the lux operon. Then, the ligation mix was electroporated into E. coli TOP10 (Invitrogen). The pGEN-lux plasmid was chosen because it contains the hok/sok genes coding for a toxin/antitoxin module, and the parR/parM partition system, both stabilizing the plasmid in the bacterial population [23]. The presence of the plasmid with the appropriate insert (pGEN-caf) was checked after plasmid extraction and digestion. The pGEN-caf construct was introduced by electroporation into Y. pseudotuberculosis V674 and recombinant strains selected. The sequence of the cloned caf1 gene was verified by sequencing. To determine the presence of a capsule, bacteria in India ink [24] were examined by phase-contrast microscopy. To quantify the production of the F1, ELISA plates (NUNC) were coated with the anti-F1 mAb G5-18 [25], followed by a 1% BSA blocking solution in PBS. Serial dilutions of bacterial suspensions (109 to 104 cfu/ml) in PBS containing 0. 1% BSA were laid in the wells. The plate was centrifuged for 10 min (1000 g) prior to incubation for 1 h at room temperature. After 3 washes with PBS, biotinylated G5-18 anti-F1 mAb (1 µg/ml) was incubated for 1 h, followed by 30 min with Streptavidin coupled to horseradish peroxidase (Jackson Immunoresearch), and colorimetric revelation using TMB (OptiEIA, BD-biosciences), as previously described [17]. Mouse infections were performed in a BSL3 animal facility. Bacterial suspensions of bacteria (200 µl in saline) were given intragastrically to seven weeks old OF1 female mice (Charles River France) using a curved feeding needle. To determine the 50% lethal dose (LD50), mice (six per dose) were infected with 10 fold serial dilutions of bacterial suspensions and were monitored for 3 weeks. The method of Reed and Muench was used to calculate LD50 values [26]. In vivo dissemination was examined five or fifteen days after oral inoculation of bacteria. Peyer' s patches, spleen and feces (two fecal pellets from the large intestine) were collected aseptically from euthanized mice and were homogenized in sterile PBS using 3 mm glass beads and an electric mill (TissueLyser®, Qiagen). The bacterial load was determined by plating serial dilutions of the homogenates. The severity of lesions caused to tissues by Y. pseudotuberculosis strains was analyzed histologically. Animals were euthanized and target organs were fixed with 4% buffered formaldehyde for 48 h, embedded in paraffin, cut in 5 micrometers sections, and stained with hematoxylin–eosin. Histological sections were read blindly and lesions were quantified using a previously described scale [17] ranging from 0 to 10. For immunohistological detection of the F1 antigen produced in vivo, Peyer' s patches collected 5 days after vaccination with the V674pF1 strain (1010 cfu) were fixed and embedded in low-melting point paraffin (polyEthylene Glycol distearate, Aldrich). Endogenous peroxidase activity was eliminated after deparaffinization by incubation in 0. 3% hydrogen peroxide for 30 min and non-specific binding sites was blocked for 30 min, prior to incubation (1 h) with the biotinylated anti-F1 G5-18 mAb. As a specificity control, an irrelevant and isotype-matched biotinylated mAb was used. After washes in PBS, sections were incubated for 1 h with Histofine® Simple Stain MAX PO (Rabbit specific; Nichirei corp.) and peroxidase activity was detected using 3-amino ethylcarbazole (AEC) substrate (Sigma). Tissues counterstained with Harris' hematoxylin were then observed using a Nikon Eclipse microscope. Blood from live animals was collected by puncture of the maxillary artery with a Goldenrod lancet (Medipoint, USA). To perform intestinal lavages, the gut section extending from the stomach to the cecum was cut from euthanized mice and flushed with 10 ml cold PBS containing protease inhibitors (Complete® from Roche plus 10 mM PMSF from Sigma). After centrifugation (10 min at 10. 000 rpm), supernatants were collected and all samples were frozen until use. Bronchoalveolar lavages were performed by injection of PBS containing protease inhibitors in the cannulated trachea. To quantify IgG and IgA specific for Yersinia antigens by ELISA, microtiter plates (NUNC) were coated with 5 µg/ml of a sonicate of Y. pseudotuberculosis IP32953 (hereafter referred to as Y. ptb Ag.) grown at 37°C on LB agar, as described before [17]. To quantify F1–specific IgG and IgA, plates were coated with F1 antigen (10 µg/ml), as described previously [25]. After blocking plates with 5% dry milk in PBS containing 0. 1% Tween 20, bacteria grown at 37°C were serially diluted in PBS containing 0. 1% BSA and were incubated in wells. Bound antibodies were detected using horseradish peroxidase (HRPO) –coupled rat antibodies specific for mouse IgG (Becton-Dickinson Pharmingen) or IgA (Caltag Laboratories), and HRPO activity was revealed using TMB substrate (OptiEIA, BD Pharmingen). Antibody titers were calculated as the reciprocal of the lowest sample dilution giving a signal equal to two times the background. Spleens taken aseptically from euthanized animals were cut in pieces and dissociated using cell strainers (BD Biosciences). Erythrocytes were lyzed using Gey' s hemolytic solution [27] and splenocytes were extensively washed with cold PBS. Cells resuspended in RPMI 1640+Glutamax™ (Invitrogen) supplemented with 5% fetal bovine serum, penicillin/streptomycin and 10 mM ß-mercaptoethanol were laid in 24 wells plates (5×106/well) and stimulated with either Y. ptb Ag. (5 µg/ml), the F1 antigen (5 µg/ml) or Concanavalin A (1 µg/ml; Sigma) as a positive control. After three days, the supernatant was collected and the cytokine content was determined using IFNγ and IL-17 assays (Duosets, R&D Systems). The fully virulent Y. pestis strain CO92 or its non-encapsulated derivative CO92Δcaf1 (Table 1) were grown at 28°C and suspensions in saline containing 105 or 107 cfu (33×LD50 or 3,300×LD50, respectively) were prepared. Mice vaccinated or not 28 days before were anesthetized and were infected by instillation of 10 µl of Y. pestis suspensions in the nostrils. Animal survival was monitored for 21 days. The Log-rank (Mantel-Cox) test was used to compare survival curves (protection). The non-parametric Mann-Whitney test was used to compare lesions, weight, antibody titers and cytokines production. Strain IP32953 was chosen to generate an irreversibly attenuated Y. pseudotuberculosis strain because its genome has been determined [20]. The HPI, encoding the Yersiniabactin iron capture system [28], was deleted by allelic exchange, generating IP32953ΔHPI (Table 1). The LD50 of IP32953ΔHPI (LD50 oral = 6. 8×108) was 16 times higher than that of the parental IP32953 (4. 2×107 cfu). Since more attenuation was required for a vaccine, the chromosomal psaA gene encoding the pH 6 Ag pilus [29]) and the virulence plasmid-borne yopK gene, encoding the type three secretion system effector protein YopK [30], were additionally deleted. The triple deletant (ΔHPI, ΔPsaA, ΔYopK) generated, named V674, exhibited a strong virulence attenuation (LD50>3×109 cfu), which was comparable to that of strain IP32680 [17]. Mice receiving 108 cfu of V674 presented no signs of disease and no weight loss (Fig. S1A), whereas the parental IP32953 induces severe signs of disease and weight loss before death [17]. Vaccination with a single oral dose (108 cfu) of V674 conferred protection to 69% of mice against an intranasal challenge with the fully virulent Y. pestis strain CO92 at a dose lethal for naive animals (105 cfu = 33×LD50). Although this level of protection was superior to that of IP32680 (30%; [17] it was considered insufficient, and V674 was further modified. In order to increase the ability of the candidate vaccine strain to induce a protective immunity against Y. pestis, V674 was further engineered to produce the Y. pestis F1 capsule. The caf operon from CO92, required for surface production of the F1 antigen in Y. pestis, was cloned into the pGEN plasmid. The resulting pGEN-caf plasmid was introduced into V674, generating V674pF1 (Table 1). The formation of a capsule around V674pF1 bacterial cells was observed (Fig. 1A c&d). When measured using an F1-specific ELISA, F1 production by V674pF1 was identical to that of Y. pestis CO92 (Fig. 1B). To determine whether the F1 capsule was produced in vivo, immunohistological staining of F1 was performed on sections of Peyer' s patches taken from mice having received V674pF1 five days before. Small foci of F1-positive bacteria were detected using an anti-F1 monoclonal antibody (Fig. 1C), while no staining was detected when an irrelevant and isotype-matched biotinylated mAb was used as control (data not shown). The V674pF1 was thus able to produce the F1 capsule within mouse tissues. To determine whether the production of F1 had an impact on the virulence of the vaccine strain, graded doses of bacteria were inoculated orally to mice. Mice having received V674pF1 (108 or 109 cfu) presented no signs of disease and no weight loss (Fig. S1B) and no lethality was observed. A high dose of 2×1010 cfu also caused no lethality, revealing a very strong attenuation of virulence. The ability of V674pF1 to persist in the intestinal tract after oral inoculation was examined by counting bacteria present in feces (Fig. 2). At the vaccine dose of 108 cfu, amounts of V674pF1 found at day 11 (d11) were comparable to those previously noted for the virulent IP32953 (#105 cfu; [17], and were also comparable to those found with the non-encapsulated V674, indicating that attenuation of virulence or F1 production did not affect the ability of V674pF1 to colonize the gut. Levels of V674pF1 were comparable when a higher vaccine dose (109 cfu) was used. The bacteria could be detected in feces for at least 20 days post infection, although at decreasing levels as compared to d11. To evaluate the dissemination of V674pF1 toward internal organs, its presence in Peyer' s patches and spleen was examined (Fig. 3A). In Peyer' s patches, amounts of V674pF1, or V674 were similar on D5, indicating that the production of F1 did not modify its ability to infect this lymphoid tissue. Similar loads were observed with a higher V674pF1 dose (109 cfu). Ten days later (D15), lower levels of V674pF1 were observed, indicating a progressive lessening of infection, in agreement with counts seen in feces. Because animals infected by IP32953 died before D15, they could not be compared. In a deep organ such as the spleen, amounts of attenuated V674pF1 or V674 found 5 days after inoculation were significantly low as compared to IP32953, in agreement with their attenuation of virulence (Fig. 3A). Again, neither the presence of F1 nor the dose of V674pF1 used affected the splenic load. Ten days later, V674pF1 or V674 were most often not detectable, showing that the attenuated bacteria were rapidly cleared (Fig. 3A). The potential development of lesions induced by the bacteria in these target organs was examined. As a reference, the wild type IP32953 at the 108 cfu lethal dose caused severe lesions to both the spleen and Peyer' s patches (abscesses, necrosis), together with signs of erosion of the flanking intestinal mucosa (scores >6/10; Fig. 3 B&C). In contrast, the same dose of V674 strain caused mainly congestion and microhemorrhages in the spleen and liver, and infrequent abscesses (scores <2/10, Fig. 3 B) in all three tissues tested. When used at a high dose of 109 cfu to maximally reveal potential harmful effects, theV674pF1 strain also caused mild tissue lesions (scores ≤1/10; Fig. 3 B&C) that were not significantly different from those caused by V674, in spite of the dose difference. In agreement with bacterial clearance, histology of the spleen and Peyer' s patches of mice vaccinated with either attenuated strain was normal or almost normal on Day 15 (score <1; Fig. 3 B), showing that tissues had healed. Altogether, our observations demonstrate that V674pF1 inoculated orally is able to colonize the gut and to interact with Peyer' s patches, but fails to disseminate to high levels in the spleen, confirming its very high attenuation. High levels of serum IgG directed against Y. pseudotuberculosis antigens were detected in sera from mice having received one oral dose of V674pF1 (108 cfu) 21 days before but not in sera from naive mice (Fig. 4A). Increasing the vaccine dose to 109 cfu did not significantly increase IgG. Comparison with mice vaccinated with the non-encapsulated V674 strain revealed comparable anti-Yersinia IgG levels (Fig. 4A). High levels of IgG directed against F1 were detected in sera from mice vaccinated with V674pF1, and not in sera from unvaccinated or V674-vaccinated mice. Because immunization through the oral route was expected to induce a mucosal type of immune response, IgA directed against both F1 and other Yersinia antigens were measured in mucosal tissues and blood after vaccination with V674pF1. Significant amounts of IgA were detectable in both intestinal lavages and sera but not in bronchoalveolar lavages (Fig. 4 C&D). Increasing the vaccine dose from 108 cfu to 109 cfu did not significantly affect the levels of IgA observed. The ability of V674pF1 to induce a cellular immune response was evaluated by comparing cytokine production by splenocytes taken from animals vaccinated with V674pF1 or V674 (both at 108 cfu) 21 days before, or not vaccinated. Splenocytes were stimulated with either a Y. pseudotuberculosis antigenic preparation obtained by sonication, or purified F1 antigen. The mitogen ConA was used as a positive control (Fig. 5 A&B). Cells from mice vaccinated with either V674pF1 or V674 significantly produced IFNγ in response to Y. pseudotuberculosis antigens, whereas cells from control naive mice did not (Fig. 5A), revealing an antigen-specific memory response. Comparable levels of IFNγ for the V674pF1 and V674 groups indicated that the presence of F1 in V674pF1 did not affect the development of the cellular response against other Yersinia antigens. Cells from V674pF1–vaccinated mice also produced IFNγ in response to F1, whereas cells from naive or V674-vaccinated mice did not, indicating a F1-specific cellular response. IL-17 production was also examined because IL-17-producing T lymphocytes (Th17 cells) are key players of antibacterial mucosal immunity [31], [32], [33], [34]. Splenocytes from V674pF1- and V674-vaccinated mice produced IL-17 upon stimulation with Y. pseudotuberculosis antigens (Fig. 5B), whereas cells from naive mice did not, indicating the recruitment of Y. pseudotuberculosis–specific IL-17 producing cells. Because splenocytes from only half of the mice vaccinated with V674pF1 produced IL-17 upon stimulation by F1, the mean production was not statistically significant. Notably, Y. pseudotuberculosis antigens induced a stronger production of IFNγ and IL-17 than F1 (×15 and ×6 respectively), indicating that the multiple antigens included are important targets of cell-mediated immunity. The ability of V674pF1 to confer protective immunity was evaluated by challenging immunized mice intranasally with a lethal dose of the fully virulent Y. pestis CO92 (105 cfu = 33×LD50). While all unvaccinated animals died within 3–4 days (Fig. 6A), a single oral inoculation of V674pF1 (108 cfu) resulted in complete (100%) protection. In contrast, vaccination with V674 conferred protection to 69% of animals only. When a very high challenge dose of 107 cfu CO92 (3,300×LD50) was used to mimic a severe contamination, mice vaccinated with 108 cfu of V674pF1 showed 80% protection and this protection reached 94% when a vaccine dose of 109 cfu was administered (Fig. 6B). Finally, we evaluated whether the immunity induced by V674pF1 was protective against a virulent F1-negative Y. pestis. To this aim, a CO92Δcaf1 Y. pestis was constructed. This mutant had an LD50 comparable to that of CO92 by the intranasal route (LD50 = 5. 6×103 cfu as compared to 2. 8×103 cfu for CO92). Mice vaccinated orally with 109 cfu of V674pF1 completely resisted a challenge infection (100% survival) by CO92Δcaf at both the 105 cfu infectious dose and the high 107 cfu dose (Fig. 6C). Our initial study demonstrated that the naturally attenuated Y. pseudotuberculosis strain IP32680 could serve as an oral vaccine against bubonic plague [17]. However, this strain had weaknesses that precluded its development for human use. Because its genome had not been analyzed, its naturally low virulence could have resulted from a point mutation, with the possibility that a reverse mutation could restore virulence. In addition, IP32680 inoculated orally conferred only a weak level of protection (30%) against the pneumonic form of plague [17], after two vaccine doses. The goals of the present work were therefore twofold: first, to obtain a strain whose attenuation was irreversible and well characterized, and second to provide high-level protection against pneumonic plague. The first objective was reached by using strain IP32953, whose genome has been sequenced [35], and to attenuate it by deleting three major virulence genes, thus preventing reversion to virulence. The genetically attenuated Y. pseudotuberculosis V674 strain provided 69% protection against pneumonic plague, and therefore was clearly more efficient than our previous IP32680 strain. However, it was still less efficient than attenuated Y. pestis strains used as plague vaccines in the past such as EV76 [36] or recently proposed engineered strains [37], [38]. Since the pioneer studies of Meyer, the F1 antigen composing the Y. pestis capsule is recognized as a major target of protective immunity due to its abundance and easy access at the bacteria surface [39]. F1 is absent from Y. pseudotuberculosis, and to enhance the efficiency of V674, the second step of our work was to introduce the caf operon coding for F1 into V674. With the presence of F1 at its surface, V674pF1 conferred full protection (100%) against pneumonic plague. Even against a very high intranasal challenge dose of CO92 (107 cfu, i. e. 3,300×LD50), V674pF1 had an excellent protective score (94%). V674pF1, after a single oral dose, thus reached a level of protection that had, to our knowledge, not been reported for other live vaccines, including attenuated Y. pestis strains [37], [40], [41] or recombinant vectors producing Y. pestis antigens such as Salmonella [11], [42] or viruses [12], [43]. The F1 capsule is not an essential virulence factor for Y. pestis in mammals (reviewed by [44]), as shown by the fact that F1-negative Y. pestis mutants are still mortal for mice, primates and humans ([45], [46], the present work) although it is required to achieve full pathogenicity in certain mouse strains [47]. Y. pestis virulence is recognized to be multifactorial, so that the transfer of a single gene in an avirulent Y. pseudotuberculosis was unlikely to increase its virulence. Indeed, high oral doses of V674pF1 induced no lethality, in agreement with similar observations in F1-producing Salmonella candidate vaccines [11]. The live attenuated vaccines have several advantages over subunit vaccines. Recently developed candidate vaccines against plague are composed of two antigens: the Y. pestis-specific capsular F1 antigen and the virulence plasmid-encoded V antigen (LcrV) common to the three pathogenic Yersinia species. Molecular and live vaccines based on F1 and V provide protection to mice against pneumonic plague [12], [13], [14], [16], however they confer only variable levels of protection to non-human primates [48]. Such molecular vaccines using Alum as adjuvant mainly induce antibody production against the F1 antigen [49], allowing virulent Y. pestis variants lacking the F1 antigen to escape from the protective immunity of anti-F1 antibodies [46]. In contrast, live vaccines are strong inducers not only of humoral immunity but also of cell-mediated immunity [48], an important component of protection against pneumonic plague [50]. We observed that splenocytes from mice orally vaccinated with both V674pF1 and V674 strongly produced IFNγ upon restimulation in vitro with Y. pseudotuberculosis antigens, indicating the development of a Y. pseudotuberculosis–specific cellular immunity. IFNγ typically characterizes the type 1 response critical for optimal vaccine-induced protection against Y. pestis infection. Indeed, it was previously shown that injection of IFNγ and TNFα protects mice against Y. pestis infection [51] and that neutralization of these cytokines abrogates vaccine-induced protection against pneumonic plague [52]. IFNγ activates phagocytes and help them destroy internalized bacteria. Therefore the potent IFNγ production by splenocytes from vaccinated mice observed in the present work may have such a role. The recruitment of Yersinia-specific cells producing IL-17 (Th17) was also observed. There is growing evidence that the development of Th17 cells is critical to vaccine-induced protection against mucosal infections by pathogenic bacteria, parasites, viruses and fungi [31], [53]. Indeed, IL-17 is a powerful inducer of PMN recruitment and release of antimicrobial peptides, and contributes to immunity induced against pneumonic plague by an attenuated Y. pestis candidate vaccine [34]. Because such effector Th17 cells can collaborate with Th1 lymphocytes [53], the induction of both subsets by V674pF1 may be a key of the high protection observed against Y. pestis in the lungs. It is most notable that vaccination using the live attenuated V674pF1 Y. pseudotuberculosis strain provided full protection against pneumonic plague caused by a virulent Y. pestis strains lacking the F1 antigen, whereas the live attenuated Y. pestis KIM D27 (Δpgm), used as vaccine in other studies, failed to protect [46], [49]. This inability of a live Y. pestis to protect was interpreted as resulting from a focalization of the immune response against the abundant F1 covering the bacteria, to the detriment of other antigens [49]. On the contrary, we show here that V674pF1 was able to trigger immunity simultaneously against F1 and the large array of target antigens common to Y. pestis and Y. pseudotuberculosis, as demonstrated by the comparable IgG titers against these antigens observed after vaccination with vaccine strains producing F1 or not. Moreover, the cellular response against Yersinia antigens was not only comparable after vaccination with V674 or V674pF1, but was also much stronger than that induced by the F1 antigen. This absence of focalization of the immune response on F1 thereby greatly enhances the likelihood of protection against a wide spectrum of Y. pestis variants. The contrast with studies using KIM D27 as vaccine [46], [49] cannot be ascribed to a difference in the amount of F1 capsule because V674pF1 produces as much F1 as Y. pestis. It could rather result from yet unidentified differences of immunogenicity between the two Yersinia species, or on the different routes of vaccination (intramuscular versus oral). In this regard, the F1 capsule also did not alter the capacity of the Y. pseudotuberculosis strain to settle in the intestinal tract. This was however not surprising because Y. pestis is virulent by the oral route, as shown by human plague cases after eating meat from an infected animal [54]. That V674pF1 was efficient through the oral route was an additional advantage because oral vaccination is both convenient, well accepted by persons, and avoids the risk of contamination through used syringes during mass vaccination. In previous studies of mouse vaccination with F1-V subunit vaccines, repeated injections were required to obtain full protection [14], [16]. In contrast, full protection against pneumonic plague was obtained in the present work after vaccination with a single oral dose of V674pF1. The capacity of live vaccines to stimulate immunity for an extended period of time was the likely key to this efficiency. V674pF1 given by the oral route persisted for weeks in the gut, allowing a prolonged antigen presentation to the immune system. F1 production significantly improved the performance of V674pF1 compared to V674, consistent with the development of F1-targeted effector mechanisms efficient in the lungs. The production of high amounts of IgG and IgA indicated that the humoral immune response was triggered at both systemic and mucosal levels. Antibodies contribute to defense against pneumonic plague, as shown by previous studies in which non-immune or immunodeficient mice were protected by instillation of anti-F1 antibodies in the airways [55], [56], [57]. Seric IgG have an easy access to the highly vascularized lung tissues and those induced by V674pF1 may have played such a role. Because immunization started at a mucosal surface, IgA could also have been actors of V674pF1-induced lung immunity [58]. No IgA could however be detected in bronchoalveolar lavages, showing that IgA were not necessary to protection. In summary, this study demonstrated that a high level protection against pneumonic plague can be obtained by a single oral vaccination with the live attenuated Y. pseudotuberculosis V674pF1 producing the Y. pestis F1 capsule. Because the strain has been irreversibly attenuated by deletion of essential virulence factors, it colonizes the intestinal tract without causing lesions and stimulates both humoral and cell-mediated anti-plague immunity. Easy to administrate orally and costless to produce, this candidate vaccine is therefore well adapted to mass vaccination in endemic tropical regions, offering promising perspectives to control pneumonic plague mortality and transmission. | Title: An Encapsulated Yersinia pseudotuberculosis Is a Highly Efficient Vaccine against Pneumonic Plague Summary: Plague, among the most deadly infections of mankind' s history, is present in Africa, Asia and America, and is currently re-emerging, recently causing cases in areas from where it had disappeared for decades. Pneumonic plague, its most deadly and contagious form, is responsible for human-to-human spreading of the infection. Vaccination would be an effective means to control the disease, but no efficient vaccine is currently available. Because live vaccines are potent inducers of protective immunity, our strategy was to use a Yersinia pseudotuberculosis, closely related to Y. pestis but genetically more stable, to make it suitable for use as live oral vaccine. We have developed a genetically defined Y. pseudotuberculosis strain strongly attenuated by deletion of virulence factors genes, which was also induced to produce the Y. pestis F1 pseudocapsule. A single oral dose was harmless and provided high- level protection against pneumonic plague. Such a candidate vaccine offers promising perspectives to control pneumonic plague mortality and transmission. | 9,541 | 260 | lay_plos | en |
Write a title and summarize: Root-knot nematodes (RKN) are obligatory plant parasitic worms that establish and maintain an intimate relationship with their host plants. During a compatible interaction, RKN induce the redifferentiation of root cells into multinucleate and hypertrophied giant cells essential for nematode growth and reproduction. These metabolically active feeding cells constitute the exclusive source of nutrients for the nematode. Detailed analysis of glutathione (GSH) and homoglutathione (hGSH) metabolism demonstrated the importance of these compounds for the success of nematode infection in Medicago truncatula. We reported quantification of GSH and hGSH and gene expression analysis showing that (h) GSH metabolism in neoformed gall organs differs from that in uninfected roots. Depletion of (h) GSH content impaired nematode egg mass formation and modified the sex ratio. In addition, gene expression and metabolomic analyses showed a substantial modification of starch and γ-aminobutyrate metabolism and of malate and glucose content in (h) GSH-depleted galls. Interestingly, these modifications did not occur in (h) GSH-depleted roots. These various results suggest that (h) GSH have a key role in the regulation of giant cell metabolism. The discovery of these specific plant regulatory elements could lead to the development of new pest management strategies against nematodes. Glutathione (GSH) is a tripeptide, γ-glutamyl-cysteinyl-glycine, present in a wide range of organisms. It is a low molecular weight thiol which in plants is involved in antioxidant defense, detoxification of xenobiotics and tolerance to abiotic and biotic stresses [1]. GSH regulates the expression of stress defense genes and is involved in plant resistance to oomycete and bacterial pathogens and insect herbivores [2]–[4]. GSH is also involved in organ development and its role in seed maturation and root and leaf growth has been established [5]–[7]. In certain legumes, a GSH homolog, homoglutathione (hGSH), is also present instead of, or in addition to, GSH [8]–[10]. The synthesis of GSH is a two-step process. In the first step, γ-glutamylcysteine synthetase (γ-ECS) produces the dipeptide γ-glutamylcysteine (γ-EC) from L-glutamic acid and L-cysteine and regulates the accumulation of GSH and hGSH [ (h) GSH]. The formation of GSH and hGSH is determined by the substrate specificity of the enzyme catalyzing the second step. Glutathione synthetase (GSHS) catalyses the addition of glycine to γ-EC, whereas homoglutathione synthetase (hGSHS) catalyses the addition of β-alanine to γ-EC. In the model legume Medicago truncatula, we have shown that (h) GSH deficiency alters the nitrogen fixing symbiotic interaction and reduces the formation of root nodules [11]. The transcriptomic response of (h) GSH-deficient plants to Sinorhizobium meliloti infection showed a downregulation of genes involved in meristem formation and an increased expression of several genes involved in the early plant defense reaction against abiotic or biotic stresses [12]. Thus (h) GSH may regulate both nodule neoformation and the plant defense response during symbiosis [12]. Plant-parasitic nematodes that infect M. truncatula and other legumes have emerged as models for studying the molecular dialogue during plant-nematode interactions and investigating whether beneficial plant symbionts and biotrophic pathogens induce distinct or overlapping regulatory pathways [13]–[17]. Root-knot nematodes (RKN, Meloidogyne spp.) are obligate root pathogens that interact with their hosts in a remarkable manner. During a compatible interaction, infective second stage RKN juvenile (J2) migrate intercellularly towards the vascular cylinder and induce the redifferentiation of root cells into specialized nematode feeding cells named giant cells (GCs). GCs are hypertrophied and multinucleate. They are the result of successive nuclear division without cell division and isotropic growth [18]. Mature GCs are metabolically very active, and act as transfer cells between vascular tissues and RKN. They are the sole source of nutrients for the feeding nematode and are thus essential for nematode growth and development [19]. Hyperplasia of neighboring cells (NCs) leads to the gall, the characteristic symptom of RKN infection. Once sedentarized, J2 molt three times to reach the adult stage. The reproduction of M. incognita is parthenogenetic: males migrate from the root and are not required for reproduction whereas the pear-shaped females produce and extrude eggs in a gelatinous matrix onto the root surface. The formation of both nodule and gall requires root cell dedifferentiation and modification of their cell cycle [20], [21]. Moreover, both nematodes and rhizobia seem to actively modulate the host plant defense, so as to allow the compatible interaction [22], [23]. The modifications to the plant defense and organogenesis observed in these plant-microbe interactions led us to analyze (h) GSH metabolism in galls. We studied the involvement of these tripeptides in the M. incognita development cycle in M. truncatula and tested for modifications of gall metabolism under (h) GSH deficiency. The development cycle of M. incognita in M. truncatula is 6–7 weeks long. We analyzed (h) GSH metabolism during the RKN life cycle. First, the expression of M. truncatula γECS, GSHS and hGSHS genes was evaluated by qRT-PCR (Figure 1A). The expression of γECS and hGSHS was significantly lower in galls than in uninfected roots from 2 wpi (Figure 1B and D). In contrast, no significant difference in the expression of GSHS was observed between galls and uninfected roots (Figure 1C). We tested whether the changes in the expression of the genes involved in (h) GSH synthesis correlated with the GSH and hGSH pools (Figure 2A). The quantification of (h) GSH pools by HPLC analysis (Figure 2) showed that hGSH was significantly less abundant in galls than in uninfected roots during the first two wpi corresponding to the period of GC formation (Figure 2A). By contrast, the GSH pool was significantly larger in galls than in uninfected roots 3 and 5 weeks post infection (wpi) with 4 fold-higher level in mature galls 5 wpi (Figure 2B). To assess the involvement of (h) GSH in the plant-nematode interaction, we analyzed the production of egg masses by the nematode in (h) GSH-depleted plants. The plant (h) GSH pool was depleted pharmacologically with L-buthionine-[S–R]-sulfoximine (BSO), a specific inhibitor of (h) GSH synthesis. The effect of BSO treatment on nematode fitness was analyzed by treatment with 1 mM BSO supplemented with 1% resorcinol, a compound shown to induce solute uptake in nematodes [24]. No difference in nematode reproduction was observed between BSO-treated nematodes and controls (Figure S1). Treatment with 0. 1 mM BSO applied one week before infection led to an 85% reduction of total (h) GSH in roots as previously described [11]. The primary root of each control and (h) GSH-depleted plants was then inoculated with M. incognita and the production of egg masses at 7 wpi was used as a measure of nematode reproduction efficiency (Figure 3). A mean of 23 egg masses was produced in control plants at 7 wpi (Figure 3A). BSO treatment led to a 75% reduction in the (h) GSH content and a 95% diminution of egg mass production in (h) GSH-depleted plants relative to control plants. To verify that this reduction in egg mass production was related to the decrease in (h) GSH content and not to another secondary effect of BSO, RNAi was used to deplete (h) GSH in M. truncatula roots of composite plants [25]. The transgenic roots carrying the γecs-RNAi construct were compared with transgenic roots expressing an RNAi construct for the Green Fluorescent Protein (GFP) as a control. The number of egg masses and the (h) GSH content of composite plants were analyzed for each individual root at 7 wpi (Figure 3B). Both (h) GSH content and the number of egg masses were significantly lower in the γecs-RNAi roots than the control gfp-RNAi plants. These experiments demonstrate that the reduction in (h) GSH content in galls correlates with a decrease in nematode egg mass production. As pharmacological and genetic (h) GSH depletion resulted in similarly impaired nematode reproduction, we mainly used BSO treatment to produce sufficient amounts of homogeneous material for further experiments. However, the major results of (h) GSH depletion were confirmed on genetically-modified material. To determine whether the reduction of egg masses was linked to a delay or an arrest in nematode development, galls were dissected at 4 and 7 wpi and the number of nematodes at each developmental stage (juvenile, male and female) was counted (Figure 4). At 4 wpi, an average number of 27 nematodes were detected per control plant whereas only 18 nematodes were observed in each (h) GSH-depleted root, suggesting that nematode infection is affected by (h) GSH depletion. Thirteen of the nematodes were at the female stage in control galls. In contrast, no female was observed in (h) GSH-depleted galls: almost all nematodes were at the juvenile stage and few males were identified in (h) GSH-depleted galls (Figure 4). Under genetic (h) GSH depletion, a significant lower number of nematodes at the female stage was also observed in the γecs-RNAi roots than in the gfp-RNAi control ones (Figure S2A). Moreover, the proportion of males was also significantly increased in γecs-RNAi roots (Figure S2B). At 7 wpi, the proportion of females was much lower in (h) GSH-depleted galls (23%) than in control galls (90%) and more than half of the nematodes were still juvenile; (h) GSH-depleted galls also contained a large proportion of males (23% vs 0. 1% in controls) (Figure 4). Thus, the (h) GSH depletion substantially reduced the number of females per gall (from 27 for controls to 2. 3 for (h) GSH-depleted galls) consistent with its effects on egg mass formation. In addition, the numbers of nematodes in (h) GSH-depleted galls at 7 wpi shows that juveniles present at 4 wpi mostly molted into males, or were eliminated from the gall. The dissection analysis showed that nematode development was impaired at four wpi as no female was detected in (h) GSH-depleted plants at this time point. To test the effect of (h) GSH deficiency on the formation of GC, molecular and cellular analyses were performed at 2 wpi (Figure 5). First, the expression of marker genes involved in GC development was evaluated by qRT-PCR (Figure 5A). The establishment of RKN infestation is associated with the suppression of plant defense responses and the induction of genes encoding proteins involved in both cell wall and DNA metabolism [22]. We therefore studied the expression of the defense-related genes, Pathogenesis-Related 1 protein and patatin, of expansin and of histone H3. During the interaction between M. truncatula and M. incognita, the expression of both Pathogenesis-Related 1 protein and patatin was significantly weaker in galls at 2 wpi than in uninfected controls; however, the expression of both expansin and histone was higher in galls than controls. No significant difference was observed between the expression of these four marker genes in (h) GSH-depleted galls and that in the controls. GC morphology was analyzed to detect potential morphological effects (Figures 5B and 5C). Microscopic analysis at 2 and 3 wpi revealed GCs with dense cytoplasm, multiple small vacuoles and nuclei observed in both (h) GSH-depleted galls and control galls (Figures 5B, 5C and Figure S3). Thus, there was no significant molecular or cellular difference between (h) GSH-depleted galls and control galls strongly suggesting that GC ontogenesis was unaffected by (h) GSH depletion. Depletion of (h) GSH was thus associated with impaired nematode development and in particular the absence of females at 4 wpi. We performed a metabolomic analysis of control and (h) GSH-depleted roots and galls at 3 wpi to assess primary metabolic effects. We investigated the major compounds of the primary metabolism in roots and galls through an untargeted proton Nuclear Magnetic Resonance (1H-NMR) analysis approach. Eighteen primary and secondary metabolites were identified in the 1H-NMR spectra of each extract (Figure S4) after peak assignment using chemical shift reported in the literature and metabolomic databases, with assistance from 2D NMR and/or by spiking samples with commercial compounds. Eighteen additional metabolites remained unidentified. Clear differences between uninfected roots and galls were obvious on visual inspection of spectra and were confirmed by quantification of metabolites. Eighteen of the 37 quantified metabolites were related to sugar, organic acid and amino acid metabolism (Table 1). Starch, an important sugar reservoir in nematode-induced syncytia [26], was assayed enzymatically. Principal component analysis (PCA) was used to provide an overview of sample grouping and metabolic differences between uninfected roots and galls: we used a matrix containing the data for the 18 identified and quantified polar metabolites plus starch (Figure 6). The first principal component (PC1) of the score plots (Figure 6A), explaining 56% of the total variability, clearly separated galls (on the negative side) from uninfected roots (on the positive side). The loading analysis (Figure 6B) suggested that the major metabolites contributing to this separation along PC1 were sucrose, trehalose, malate, fumarate, six amino acids (aspartate (Asp), glutamate (Glu), isoleucine (Ile), phenylalanine (Phe), tyrosine (Tyr) and valine (Val) ) and trigonelline on the negative side and glyoxylate on the positive side. PCA score plot also showed that the second principal component (PC2), explaining 17% of the total variability, clearly separated (h) GSH-depleted (on the negative side) from control galls (on the positive side) (Figure 6A). Observation of PC2 loading (Figure 6B) suggested that this separation along PC2 mainly involved γ-aminobutyrate (GABA), Ile, Val, Glu, Asp and Asn on the negative side and glucose, starch and proline-betaine on the positive side. The PCA was confirmed by univariate analyses of metabolite data (Table 1). Relative to control roots, galls exhibited a significantly higher content of starch, sugars (sucrose, glucose), organic acids (malate, fumarate) and amino acids (Phe, Tyr, Val, Glu, Asp). However, the increase in amounts of these metabolites was not related to a similar modification of the expression of primary metabolism genes, the expression of which was maintained (Sucrose synthase 1, ADP-glucose pyrophosphorylase, starch synthase, α 1–4 glucan phosphorylase, pyruvate kinase) or even decreased (cell wall-invertase, mitochondrial malate dehydrogenase, malate synthase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase) in galls (Figure 7). Interestingly, the asparagine (Asn) content of control galls was significantly lower than that of control roots (Table 1). This was related to a significant decrease in expression of the asparagine synthetase and an increase in that of asparaginase (Figure 7). Proline-betaine, production of which in plants is related to the water stress response, accumulated significantly more in galls than control roots (Table 1). Trigonelline, another aminated compound related to secondary metabolism and potentially involved in salt-stress response [27], was also more abundant in galls than control roots (Table 1). Finally, trehalose accumulation may also be related to a modification of the osmotic status in galls [28]. We used these metabolite and gene expression data to establish a metabolic pathway scaffold (sucrose and starch metabolism, glycolysis and the tricarboxylic cycle connected branch points toward organic acid and amino acid synthesis) highlighting the significant differences observed between roots and galls (Figure 8A). Generally, there is little correlation between increased accumulation of quantified metabolites and the expression of the associated primary metabolism genes. Depletion of (h) GSH modified the metabolism of roots and galls in different ways. Most metabolites in roots were not significantly modified by (h) GSH-depletion. PCA analysis of the 18 identified and quantified polar metabolites plus starch showed that (h) GSH-depleted and control uninfected roots had a similar composition of polar metabolites (Figure 6A). Significant variations were observed only for the hydrophobic amino acids Ile, Phe and Tyr, for trigonelline and for starch (Table 1). Indeed, the starch content was decreased 3-fold and starch synthase was significantly down regulated by (h) GSH depletion, suggesting that starch metabolism in roots is regulated by (h) GSH content or metabolism. Unlike the findings for uninfected roots, (h) GSH depletion had substantial effects on the metabolism of galls (Figure 6). The content of nine metabolites differed significantly between (h) GSH-depleted galls and control galls (Table 1). Starch and glucose contents were significantly lower in (h) GSH-depleted galls, whereas that of sucrose was not significantly different. With the exception of malate, the abundance of which was significantly decreased, the organic acid content of galls was not significantly affected by (h) GSH depletion. In the γecs-RNAi galls, starch, glucose and malate contents were also significantly lower than in the control gfp-RNAi galls (Table S1). Consistent with these findings, the relative expression in galls of most genes involved in the metabolism of sugars and organic acids was not significantly modified by (h) GSH depletion. The amino acids content was slightly increased by (h) GSH depletion, with the exception of Asn which was increased two-fold to the range of that found in roots (Table 1). This increase in Asn content was associated with a significant decrease in asparaginase gene expression and a two-fold increase in asparagine synthetase gene expression associated with (h) GSH depletion (Figure 7). A similar trend was also observed for proline-betaine, the content of which in (h) GSH-depleted galls was close to that in uninfected roots. Thus, our data indicate that (h) GSH depletion partially reversed the effect of nematode infection on starch, glucose and Asn metabolism, and on proline-betaine accumulation. Interestingly, GABA, a compound associated with biotic and abiotic stresses [29], was markedly more abundant in (h) GSH-depleted than control galls. The significant differences between control and (h) GSH-depleted galls are summarized in a metabolic pathway scaffold (Figure 8B). As observed for the comparison between galls and uninfected roots, differences in metabolite contents were more marked than the differences between gene expression levels. This implies post-transcriptional regulatory mechanisms, such as post-translational modifications or metabolic controls, in the metabolic modifications associated with (h) GSH depletion of galls. M. truncatula roots contain two low molecular weight thiols, GSH and hGSH [9]. The (h) GSH content is significantly higher in galls than in roots at later stages of gall functioning. Surprisingly, γECS transcript level was lower in galls than in roots whereas this gene should regulate the level of (h) GSH. The post-transcriptional regulation of γECS [33], [34] may explain the discrepancy between γECS transcript level and GSH accumulation. (h) GSH accumulation has been observed in several developmental conditions involving endoreduplication and enhanced metabolic capacity such as in symbiotic nitrogen fixation [9] and in trichomes [35], both physiological modifications also occurring during gall formation and function [18], [22], [36]. In addition, the accumulation of GSH in galls may be caused by the nematode, as a GSHS has been identified amongst the proteins secreted by M. incognita [37]. Finally, (h) GSH accumulation is also associated with the nematode secreting multiple redox- and (h) GSH-regulated proteins, including thioredoxin, glutathione peroxidases and glutathione-S-transferases, required for the completion of nematode life cycle [37], [38]. Indeed, the control of the plant cell redox status through the modification of the (h) GSH content may be a key regulator of the GC effectiveness. We show here that root (h) GSH deficiency strongly impairs nematode reproduction. This reduction of egg masses seems to be largely a consequence of the nematode sex ratio in galls. At 7 wpi, galls in (h) GSH-depleted plants harbored only one third as many nematodes as controls, suggesting that most juveniles developed into males and therefore migrated from the gall to soil such that they were not found in the gall by dissection. An hypothesis might be that BSO would be involved in direct impairment of GSH production in nematodes [39], [40] and thus modify their development and egg mass production. Analysis of M. incognita genome using Caenorhabditis elegans γECS and GSHS sequences shows that the GSH biosynthesis genes are present in M. incognita. Moreover, HPLC analysis shows that GSH is produced in M. incognita J2 larvae (unpublished data). The effect of GSH depletion on M. incognita development could not be directly tested as it is a plant obligatory parasite. However, data provided on WormBase (http: //www. wormbase. org) showed that GSH does not play a major role in both development and health in C. Elegans. GSH depletion induced by γECS-RNAi or gene deletion is not larval or embryonic lethal and does not induce slow growth and female sterility [41]–[43]. Finally, the lower egg mass production, the modifications of the nematode sex-ratio and metabolite contents observed in both BSO-treated plants and transgenic roots expressing a plant specific γECS-RNAi construct show that these modifications are not linked to direct impairment of nematode function by BSO. During symbiosis between M. truncatula and S. meliloti, the (h) GSH depletion reduces the formation of nodule meristems [11]. Transcriptomic analysis evidences the involvement of (h) GSH regulation both in plant development and defense responses [12]. In contrast, under similar conditions, the development of the feeding site was not significantly affected and the expression of defense-related and development-related genes was not modified. Therefore, M. incognita is able to manipulate plant metabolism under (h) GSH depletion to avoid the defense and developmental phenotype observed during the establishment of nitrogen-fixing symbiosis. Root and gall metabolomic profiling showed that most of the analyzed metabolites were significantly more abundant in galls than in uninfected roots. These modifications, and the analysis of the expression of numerous genes involved in primary metabolism, indicate that the gall metabolism differs substantially from that in uninfected roots. One of the striking differences concerning general metabolite accumulation is the significantly lower Asn content in galls than in roots. This, and the associated upregulation of asparaginase and the down regulation of asparagine synthase, shows that nitrogen metabolism is modified in galls. Asn is the major nitrogen transporting compound in temperate legumes such as Medicago [44], [45]. The primary site of Asn synthesis is the root and it follows that, through loading into the xylem, Asn is the principal nitrogen source for amino acids and protein synthesis in leaves. Thus, the decrease in Asn content upon nematode infection is likely to result in nitrogen deprivation for the plant. This metabolic modification may thus reduce nitrogen supply to leaves and increase carbon and nitrogen accumulation in galls. Our findings show that gall metabolism involves the fine-tuning of metabolism involving both the up regulation of some metabolic pathways and the down regulation of others, so as to enhance nutriment availability for the nematodes. Analysis of metabolite contents shows that (h) GSH depletion significantly affect gall metabolism. A significant difference in metabolite content between control and (h) GSH-depleted galls was detected for half of the metabolites quantified. In contrast, (h) GSH depletion did not significantly modify the content of the major primary metabolites in uninfected roots. This result is in agreement with our previous findings that a 85% depletion of (h) GSH does not significantly affect root growth [11]. The metabolic modifications observed in (h) GSH-depleted galls include a significant reduction of malate (40%), glucose (60%) and starch (84%). Starch accumulation during the interaction between A. thaliana and the parasitic nematode Heterodera schachtii is crucial for the nematode infection and development. It may serve as long- and short-term carbohydrate storage for the feeding needs of the parasites [26]. Glucose and malate are likely substrates and probably essential for nematode nutrition. Thus, the diminution of these three metabolites under (h) GSH depletion may impair nematode carbohydrate nutrition. The development of M. incognita juveniles into males rather than females has previously been observed under unfavorable nematode feeding conditions such as low concentrations of sucrose in the growth medium, defoliation and complete removal of the host plant above-ground parts [46]–[48]. These conditions also trigger carbon starvation of the galls. Carbohydrate nutrition deficiency has been also involved in the modification of sex ratio and development of cyst nematode [49]. The development of M. incognita juveniles into males rather than females in (h) GSH-depleted galls is similar to that observed during carbohydrate deficiency. This is consistent with (h) GSH being involved in the modulation of nematode differentiation through regulation of gall carbon metabolism. Interestingly, GABA was specifically detected in (h) GSH-depleted galls. In plants, GABA accumulates in response to abiotic and biotic stresses [50]. During biotic stress induced by invertebrate pests, GABA accumulation in plant tissues reduces the feeding capacity of the pests [51]. Strikingly, the reproduction of Meloidogyne hapla is affected by GABA accumulation: egg mass production by M. hapla infecting transgenic plants accumulating GABA is lower than that by the pests infecting control plants [52]. Thus, the accumulation of GABA in (h) GSH-depleted galls may also contribute to the altered nematode reproduction. The substantial primary metabolite modifications in (h) GSH-depleted galls with reference to control galls were not associated with corresponding modification in the expression of primary metabolism genes. This suggests that (h) GSH regulates gall metabolism at levels other than transcriptional. Redox state and GSH affect the function of many enzymes through post-translational modifications such as disulfide bond reduction and cysteine glutathionylation [53]. For instance, thioredoxins and glutaredoxins, which are involved in the formation/reduction of disulfide bonds between proteins, have been implicated in the regulation of chloroplast metabolism [54], [55]. ADP glucose pyrophosphorylase, a key enzyme in the biosynthesis of starch was also shown to be redox regulated [56], [57]. Cysteine glutathionylation is an important regulatory mechanism of photosynthetic metabolism [58]. More generally, in vivo control of many glycolytic and/or TCA cycle enzymes by disulfide-dithiol interconversions (NAD-dependent GAPDH, citrate synthase, PPi-dependent phosphofructokinase, PEPC kinase, etc) has been reported in plants [59]. Thus, a redox-based control of the gall metabolism by (h) GSH may be proposed to explain our results. We cannot exclude that GSH may also be used as a nutrient by the nematode as the GSH content was strongly increased in mature galls compared to roots. However, the impact of (h) GSH depletion on gall metabolism is not in favour of a trophic role for (h) GSH. Moreover, during nitrogen fixing symbiosis in which GSH is not used as nutrient to feed the bacteroids, modifications of the (h) GSH content affects the nitrogen-fixing capacity of the nodule also showing the regulatory role of glutathione in this interaction [60]. In conclusion, we report that (h) GSH metabolism differs between galls and uninfected roots. A deficiency in (h) GSH impairs nematode reproduction by mainly altering its sex determination. This alteration in sex ratio is associated with modifications in the gall metabolism under (h) GSH depletion which have been shown to impair nematode development. Thus, we reveal a completely new role of (h) GSH in this biotrophic interaction. Interestingly, these modifications in metabolite content do not seem to occur in (h) GSH-depleted roots suggesting that (h) GSH depletion provokes metabolism modifications specific to the gall. Therefore, the reduction of (h) GSH availability in galls is a potentially useful strategy for pest management. M. truncatula ecotype A17 was used for all the experiments. Sterilized seeds were germinated for 3 days onto 0. 4% agar at 14°C. Seedlings were plated onto modified Fahraeus medium with 2 mM nitrogen [25] with 1. 4% agar and grown for 7 days before infection. Plants were germinated in the presence or absence of 0. 1 mM L-buthionine sulfoximine (BSO). For nematode infection, 100 surface-sterilized freshly hatched M. incognita J2 larvae were added on each one week old seedling as previously described [13]. One infection per plant was performed on the primary root. For BSO treatment, nematodes were incubated for 4 hours in M9 buffer (43. 6 mM Na2HPO4,22 mM KH2PO4,2. 1 mM NaCl, 4. 7 mM NH4Cl) with 1% resorcinol and 1 mM BSO and with 1% resorcinol as control. The gall corresponds to one infection point and contains multiple GCs. After infection, plants were grown 3 weeks onto Fahraeus medium with 2 mM nitrogen in the presence or absence of BSO (0. 1 mM) in a growth chamber with a day temperature of 23°C and night temperature of 20°C and with a photo-period of 16 h. Then, plants were transferred in soil mixture (30% vermiculite-70% fine gravel) until the end of the experiment. As reference samples, uninfected, primary root fragments of similar age were collected from seedlings grown under the same conditions. For gall dissection, galls were digested in a mixture of 30% Pectinex (Novozymes, Bagsvaerd, Denmark) and 15% Celluclast BG (Novozymes, Bagsvaerd, Denmark) for 12 h, dissected and nematode development stages were analyzed under a stereomicroscope. For metabolite and gene expression analyses, biological samples of galls and roots were harvested at different time points post-infection, frozen and ground in liquid nitrogen and stored at −80°C. One biological sample was issued from 20 galls or roots from 20 plants. Thiols were extracted with perchloric acid, derivatized with monobromobimane, and quantified after separation on reverse-phase HPLC as described previously [61]. Commercial GSH (Sigma, St. Quentin, France) and γ-EC (Promochem, Molsheim, France) were used as standards. The hGSH used as a standard was synthesized by Neosystem (Strasbourg, France). Total RNA of galls and uninfected root fragments were reverse-transcribed using the OmniScript cDNA Synthesis Kit (Qiagen, Courtaboeuf, France). Quantitative PCR reactions were performed using a DNA Engine Opticon 2 Continuous Fluorescence Detection system (MJ Research, Waltham, USA) and a qPCR MasterMix Plus for SYBR green I (Eurogentec, Angers, France). In each reaction, 5 µl of 100 fold-diluted cDNA and 0. 3 µM primer (sequences used are described in Table S2) were used. The PCR conditions were 50°C for 5 min, 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 1 min. Each reaction was performed in triplicate and the results represented the mean of three independent biological experiments. The specificity of the amplification was confirmed by a single peak in a dissociation curve at the end of the PCR reaction. Data were quantified by using Opticon Monitor 2 (MJ Research, Waltham, USA) and normalized with the 2−ΔΔCT method [62]. Two constitutively expressed genes Mtc27 (TC106535) and 40S Ribosomal Protein S8 (TC100533) were the endogenous controls [63]. The use of these housekeeping genes were validated by using the GeNorm VBA applet for MS Excel which determines the most stable housekeeping genes from a set of tested genes in a given cDNA sample panel [64]. PCR reactions for each of the three biological replicates were performed in technical triplicate. The absence of genomic DNA contaminations in the RNA samples was tested by PCR analysis of all samples using oligonucleotides bordering an intron in M. truncatula GSHS gene. To generate the γ-ECS-RNAi construct, a 502-bp region was amplified from the cDNA using gene-specific primers (Supplemental Table S2 on line) and cloned into the pDONR207 vector, subcloned in pENTR4 and integrated into the RNAi vector pK7GWIWG2DII, (0) [65]containing kanamycin resistance and the p35S: eGFP for selection and screening. M. truncatula plants (A17) were transformed with A. rhizogenes containing precedent construct as described previously [25] and transformed roots were selected by resistance to kanamycin and screening of eGFP. Control plants were transformed with A. rhizogenes containing the pKGWIWG2DII, (0) vector containing an eGFP DNA fragment to rule out the potential side effects linked to plant transformation or the RNAi vector. Polar metabolites were quantified using 1H-NMR of polar extracts. For the preparation of extracts and NMR acquisition parameters, special care was taken to allow absolute quantification of individual metabolites. Briefly, polar metabolites were extracted on lyophilized powder (30 mg DW per biological replicate) with an ethanol–water series at 80°C as described previously [66]. The lyophilized extracts were titrated with KOD to pH 6 in 100 mM potassium phosphate buffer in D2O and lyophilized again. Each dried titrated extract was solubilized in 0. 5 mL D2O with (trimethylsilyl) propionic-2,2, 3,3-d4 acid (TSP) sodium salt (0. 01% final concentration) for chemical shift calibration and ethylene diamine tetraacetic acid (EDTA) disodium salt (0. 5 mM final concentration). 1H-NMR spectra were recorded at 500. 162 MHz on a Bruker Avance spectrometer (Bruker, Karlsruhe, Germany) using a 5-mm dual 13C-1H cryoprobe and an electronic reference for quantification [66]. Sixty-four scans of 32 K data points each were acquired with a 90° pulse angle, a 6000 Hz spectral width, a 2. 73 s acquisition time and a 25 s recycle delay. Preliminary data processing was conducted with TOPSPIN 1. 3 software (Bruker Biospin, Wissembourg, France). The assignments of metabolites in the NMR spectra were made by comparing the proton chemical shifts with literature [66]–[68] or metabolomic database values (MeRy-B 2009, HMDB), by comparison with spectra of authentic compounds recorded under the same solvent conditions and/or by spiking the samples. For assignment purposes, 1H-1H COSY, 1H-13C HSQC and 1H-13C HMBC 2D NMR spectra were acquired for selected samples. The metabolite concentrations were calculated using AMIX (version 3. 9. 1, Bruker) and Excel (Microsoft, Redmond, WA, USA) softwares. The metabolites were quantified using the glucose calibration curve and the proton amount corresponding to each resonance for all compounds. The metabolite concentrations were calculated from concentrations in the NMR tube and sample dry weight. The 15 1H-NMR spectra of the data set were converted into JCAMP-DX format and deposited with associated metadata into the Metabolomics Repository of Bordeaux MeRy-B (http: //www. cbib. u-bordeaux2. fr/MERYB/projects/home. php? R=0&project_id=28). To explore the metabolite multidimensional data set, we used principal component analysis (PCA) on mean-centered data scaled to unit variance (MATLAB version 7. 4. 0, the MathWorks Inc, Natick MA). Starch was recovered from the insoluble fraction of the extracts used for polar metabolite extraction after ethanol–water series at 80°C (see above, Moing et al. 2004). Insoluble residues were incubated for 1 h, at 55°C, in a 0. 5 ml reaction medium containing 0. 1 M sodium acetate, and 1. 25 mg amyloglucosidase (Sigma-Aldrich, Saint-Quentin Fallavier, France). Reaction was stopped for 5 min at 100°C. Supernatants were collected and evaporated over night under vacuum. Dry residues were taken up with 0. 5 ml of 0. 3 M Hepes, pH 7. 5, and 30 mM MgSO4. Glucose, issued from starch hydrolysis, was measured as followed: 200 to 400 µl of samples were mixed with a reaction medium containing 0. 3 M Hepes, pH 7. 5,30 mM MgSO4,2. 5 mM ATP, and 2 mM NAD. Initial OD was red at 340 nm. Next, 2 Units of both hexokinase (Sigma-Aldrich, Saint-Quentin Fallavier, France) and glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides (Sigma-Aldrich, Saint-Quentin Fallavier, France) were added, and samples were incubated for 1 h, at room temperature in the dark. Final OD was red at 340 nm. The difference between the final and initial OD was used to calculate the glucose content. Starch was expressed as nmol of glucose equivalent per dry weight unit. Malate was quantified by ionic chromatography and conductimetry. Separation was performed on an IonPac AS 11 column (4×250 mm, Dionex, Sunnyvale, CA, USA) and a IonPac AG11 guard column (4×50 mm, Dionex) with a NaOH gradient including 16% of methanol. Calibration was performed with commercial standards using gravimetric method. DNA sequences were analyzed using BLAST [69] against the databases of the NCBI (http: //blast. ncbi. nlm. nih. gov/), MtGI (http: //compbio. dfci. harvard. edu/cgi-bin/tgi/gimain. pl? gudb=medicago) and the IMGAG (http: //www. medicago. org/genome/). The accession numbers of the genes used in this study are indicated in the Supplemental Table S2. All the data presented are given as means with the standard error of three or four independent biological experiments. The significance of the results was tested using Student t-test (P value ≤0. 05). | Title: (Homo)glutathione Deficiency Impairs Root-knot Nematode Development in Medicago truncatula Summary: Parasitic nematodes are microscopic worms that cause major diseases of plants, animals and humans. During compatible interactions, root-knot nematodes (RKN) induce the formation of galls in which redifferentiation of root cells into multinucleate and hypertrophied giant cells is essential for nematode growth and reproduction. The importance of glutathione (GSH), a major antioxidant molecule involved in plant development, in plant microbe interaction and in abiotic stress response, was analyzed during the plant-RKN interaction. Our analyses demonstrated that the gall development and functioning are characterized by an adapted GSH metabolism and that depletion of GSH content impairs nematode reproduction and modified sex ratio. This phenotype is linked to specific modifications of carbon metabolism which do not occur in uninfected roots indicating a peculiar metabolism of this neoformed organ. This first metabolomic analysis during the plant-RKN interaction highlights the regulatory role played by GSH in this pathogenic interaction and completes our vision of the role of GSH during plant-pathogen interactions. RKN sex ratio modification has previously been observed under unfavorable nematode feeding conditions suggesting that the GSH-redox system could be a general sensor of gall fitness in natural conditions. | 9,733 | 296 | lay_plos | en |
Write a title and summarize: Diet plays a significant role in maintaining lifelong health. In particular, lowering the dietary protein: carbohydrate ratio can improve lifespan. This has been interpreted as a direct effect of these macronutrients on physiology. Using Drosophila melanogaster, we show that the role of protein and carbohydrate on lifespan is indirect, acting by altering the partitioning of limiting amounts of dietary sterols between reproduction and lifespan. Shorter lifespans in flies fed on high protein: carbohydrate diets can be rescued by supplementing their food with cholesterol. Not only does this fundamentally alter the way we interpret the mechanisms of lifespan extension by dietary restriction, these data highlight the important principle that life histories can be affected by nutrient-dependent trade-offs that are indirect and independent of the nutrients (often macronutrients) that are the focus of study. This brings us closer to understanding the mechanistic basis of dietary restriction. Dietary restriction, also called calorie restriction, is a moderate reduction in food intake that extends healthy lifespan across a broad range of taxa, from yeast to primates (Chapman and Partridge, 1996; Colman et al., 2009; Lin et al., 2002; McCay et al., 1935). The generality of this observation has inspired confidence that the health benefits of dietary restriction might also be employed to improve human ageing (Campisi et al., 2019). In an attempt to harness its benefits, a great deal of current research is focused on discovering the nutritional components and the molecular mechanisms that underpin the lifespan benefits of dietary restriction (López-Otín et al., 2013; Simpson et al., 2017). Our current understanding of how diet modifies lifespan has grown out of evolutionary theory and experiments using model organisms. The most prominent theoretical explanation has been the disposable soma theory, which employs resource-based trade-offs to explain how dietary restriction can benefit lifespan (Kirkwood, 1977; Shanley and Kirkwood, 2000). This theory postulates that organisms will maximise fitness by strategically partitioning limiting dietary energy either to reproduction or somatic maintenance, the latter determining lifespan. This means that longer lifespan is inevitably coupled with reduced reproduction because both traits compete for the same limiting resource. Recent experimental work across a broad range of taxa has challenged the disposable soma theory by demonstrating that reproduction and lifespan respond predominantly to the balance of dietary macronutrients, not the overall energy content of the diet (Mair et al., 2005; Lee et al., 2008; Skorupa et al., 2008; Grandison et al., 2009; Solon-Biet et al., 2014; Solon-Biet et al., 2015; Simpson et al., 2017; Regan et al., 2020). Specifically, high protein, low carbohydrate diets are consistently associated with high reproduction and short lifespan, while low-protein, high-carbohydrate diets are associated with longer lifespan and lower levels of reproduction (Piper et al., 2011; Simpson et al., 2017). These data indicate that lifespan and reproduction are not in competition for limiting energy derived from the diet, but instead are optimised at different dietary protein: carbohydrate ratios. In response to these findings, an enormous effort is now focused on uncovering how macronutrient rebalancing, in particular protein dilution, acts to improve lifespan (Blagosklonny, 2006; Blagosklonny, 2010; Moatt et al., 2020; Regan et al., 2020; Speakman, 2020). Accumulating evidence indicates that the effect is mediated by reducing signalling through the amino acid sensitive Target Of Rapamycin (TOR) pathway to enhance cellular proteostasis (Sanz et al., 2004; Ayala et al., 2007; Raubenheimer and Simpson, 2009; Simpson and Raubenheimer, 2009; Taylor and Dillin, 2011; Fanson et al., 2012; Sabatini, 2017). Although detrimental for lifespan, relatively high protein, low carbohydrate diets are beneficial for female reproduction (Chong et al., 2004; Solon-Biet et al., 2015). We have studied this closely in the fruit fly Drosophila melanogaster, where the principle driver of egg production is dietary protein (Min and Tatar, 2006; Grandison et al., 2009; Piper et al., 2017). Although protein is key, females must transfer dozens of nutrients into eggs for future embryo formation and not all of these components contribute to the flies’ decision to produce eggs (Piper et al., 2014; Mirth et al., 2019; Wu et al., 2020). This means that high protein diets might drive mothers to produce eggs at a faster rate than they can support if the diet contains insufficient levels of the other components that are required to make eggs. In this scenario, the macronutrients would have an indirect effect on lifespan by changing the availability of another limiting nutrient that is required for somatic maintenance. If true, this would move the focus of mechanistic studies away from the direct effects of protein, TOR, and proteostasis, towards some other component of nutritional physiology. Distinguishing between these possible causes of death is important since it would fundamentally change our understanding of the way diet alters lifespan. It also has the important knock-on effect that we could change the way we design diets for longer life. For instance, supplementing high protein diets with key limiting nutrients would be as beneficial as restricting dietary protein or treating with pharmacological suppressors of TOR. Of the many studies that have examined the effects of dietary protein and carbohydrate on lifespan and reproduction in Drosophila, most have done so by varying dietary yeast and sugar proportions, where yeast is the flies’ natural source of protein (Mair et al., 2005; Lee et al., 2008; Skorupa et al., 2008). However, yeast also contains all of the flies’ other essential macro and micronutrients whose relative proportions can change, and thus possibly interact with protein and carbohydrates to dictate life-history outcomes. We have previously found that depriving adult female flies of a source of sterols, an essential micronutrient for insects, imposes a minor cost on reproduction, but a substantial (>50%) cost to lifespan (Piper et al., 2014; Wu et al., 2020). These data indicate that yeast sterol levels may contribute to the effects on lifespan of protein and carbohydrate. To investigate the interactions between dietary protein, carbohydrate, and sterols systematically, we have used the design principles of the geometric framework for nutrition (Simpson and Raubenheimer, 2012; Simpson and Raubenheimer, 1993) and a completely defined (holidic) diet that allows us to control the levels of each nutrient independently of all others (Piper et al., 2014; Piper et al., 2017). These data point to an important role for sterols in determining Drosophila lifespan, which we verified to be relevant in two yeast-based media that are often used in Drosophila lifespan studies. This work is critical to identifying how diet modifies lifespan at the molecular level, and highlights a new way to think about diet design to improve healthy ageing. To examine the interactive effects of dietary protein, carbohydrate, and cholesterol on Drosophila lifespan and fecundity, we used our completely defined (holidic) diet (Piper et al., 2014) to manipulate each nutrient independently of all others. We selected dietary protein and carbohydrate concentrations that we know to elicit the full range of lifespan and fecundity responses to dietary restriction (Lee et al., 2008; Piper et al., 2014, Piper et al., 2017; Ma et al., 2020). Similar to what we and others have found previously (Mair et al., 2005; Lee et al., 2008; Grandison et al., 2009; Piper et al., 2014, Piper et al., 2017; Katewa et al., 2016), lifespan and reproduction were modified by dietary protein manipulations (Figure 1). Specifically, egg production showed a linear, positive correlation with dietary protein content (Figure 1, Supplementary file 1), while lifespan showed a peak at intermediate protein (66 d median at 10. 7 g/l), and fell away at both higher (49 d median at 33. 1 g/l) and lower (43 d median at 5. 2 g/l) concentrations (Figure 1a–b, Supplementary file 2). Thus, as is typical for dietary restriction experiments, restricting dietary protein from high to intermediate levels increased lifespan and decreased reproduction (Lee et al., 2008; Skorupa et al., 2008; Grandison et al., 2009; Katewa et al., 2016; Le Couteur et al., 2016). When increasing dietary carbohydrate against an otherwise fixed nutritional background, egg laying was suppressed in a dose-dependent fashion, but lifespan remained at its maximum level and was unchanged across all carbohydrate doses (~66 d median, Figure 1c–d). The diet with the lowest concentration of carbohydrate (5. 7 g/l), which also contained the intermediate protein level (10. 7 g/l), supported both maximum lifespan (Figure 1d; 66 d median) and the highest level of egg laying (75 eggs/female) of any diet in our experiment. Thus, as we have previously shown (Piper et al., 2017), balancing the dietary protein and carbohydrate concentrations can reveal a single dietary optimum for both traits, showing that lifespan shortening is not necessarily caused by high egg laying alone. Most dietary restriction studies on Drosophila vary dietary protein by modifying the yeast levels in food (Chapman and Partridge, 1996; Mair et al., 2005; Lee et al., 2008; Skorupa et al., 2008). While yeast is the flies’ major source of protein, it is also their only source of dozens of other nutrients, including sterols, which are essential micronutrients for insects (Carvalho et al., 2010). To quantify the effects of varying dietary sterol levels on fly lifespan and egg laying, we maintained flies on the same set of diets as above, varying in protein and carbohydrate concentrations, while also varying cholesterol across four different levels: 0 g/l, 0. 15 g/l (low), 0. 3 g/l (medium; also our standard level), and 0. 6 g/l (high). Initial analysis of the data showed that diet type, when defined by variation of macronutrient composition, as well as variation in cholesterol concentration both significantly modified egg laying and lifespan (Figure 2, Supplementary files 3 and 4). By contrast, we saw no main effect of dietary energy density (calories) on either trait, which is consistent with previous findings showing that these traits are driven by the relative proportion of protein and carbohydrate in the diet independently of caloric value (Lee et al., 2008; Mair et al., 2005; Skorupa et al., 2008). We therefore proceeded in our analysis to assess how cholesterol modified the effects of protein and carbohydrates on these traits. We first compared the flies’ responses to variation in both protein and cholesterol (Figure 2—figure supplement 1a–b). In general, lifespan was optimised at our intermediate dose of protein, while increasing cholesterol was beneficial, but with diminishing effect as its concentration was increased (Figure 2, Supplementary file 5). Interestingly, changing cholesterol modified the flies’ lifespan response to protein, an effect that can be seen when the data are separated by level of cholesterol addition (Figure 2—figure supplement 1a–b). At 0 g/l cholesterol (Figure 2a) increasing protein concentration in the diet decreased lifespan. However, at 0. 15 g/l cholesterol, the shape of the response changed such that only the highest protein concentration decreased lifespan (35 d median; Figure 2c) when compared with intermediate (9. 7 g/l; 55 d median) and low-protein (4. 7 g/l; 52 d median) diets. At 0. 3 g/l of cholesterol, lifespan was highest on the diet with intermediate protein concentration (66d median) and flies on the high protein diet were longer lived (49 d median) than the flies on the lowest protein diet (43 d median). Finally, increasing cholesterol from 0. 3 g/l to 0. 6 g/l (Figure 2g – Figure 2—figure supplement 1a–b) did not change the way that lifespan responded to protein. Thus, lowering dietary cholesterol was detrimental for lifespan and it intensified the negative effects of increasing dietary protein concentrations. Across the same set of diets, we observed a generally beneficial effect on egg laying of increasing dietary protein and cholesterol, and both had diminishing benefits as their concentrations increased (Figure 2—figure supplement 1d–e, Supplementary file 6). Cholesterol also modified the way egg laying was affected by dietary protein (Figure 2). Increasing cholesterol from 0 g/l (Figure 2a) to 0. 15 g/l (Figure 2c) amplified the positive effect on egg laying of increasing dietary protein. Further increasing cholesterol to 0. 3 g/l had an additional benefit for egg laying (Figure 2e), but only for flies on the highest protein diet (compare Figure 2c with Figure 2e), while increasing cholesterol even further, to 0. 6 g/l (Figure 2g), did not change egg laying from that seen on 0. 3 g/l. Thus, the response of egg laying to increasing protein was only compromised when cholesterol was completely removed from the diet, or when cholesterol was low (0. 15 g/l) and protein was high (30 g/l) (Figure 2c). Together, these data show that reducing cholesterol had negative effects on both lifespan and egg laying, and that these negative effects became more pronounced with increasing dietary protein. Furthermore, the negative interaction between lowering cholesterol and increasing protein was more severe and occurred at a lower protein concentration for lifespan than it did for egg laying. Next, we looked to see if changing dietary cholesterol modified the responses of lifespan and egg laying to variation in carbohydrate concentration (Figure 2b, d, f, h - Figure 2—figure supplement 1c and f Supplementary files 5 and 6). At 0 g/l cholesterol, lifespan was generally short (31d median) but positively affected by increasing dietary carbohydrate (up to 40 d median) (Figure 2b). As dietary cholesterol was increased to 0. 15 g/l, lifespan on all diets was higher and the positive effect of increasing carbohydrate was preserved (Figure 2d). However, when cholesterol reached 0. 3 g/l, the flies were constantly long-lived, and lifespan was unaffected by dietary carbohydrate level (66 d median) (Figure 2f). This pattern was not changed by increasing cholesterol further to 0. 6 g/l (Figure 2h). Thus, each of our dietary carbohydrate levels could support maximal fly lifespan, but the lower carbohydrate diets were more susceptible to the detrimental effects of dietary cholesterol dilution. Increasing dietary carbohydrate had a generally negative impact on egg laying, and this effect was modified by the benefits of increasing dietary cholesterol (Figure 2—figure supplement 1f, Supplementary file 6). Without any cholesterol in the food, egg laying was consistently low and was negatively affected by increasing dietary carbohydrate (Figure 2b). This negative effect of carbohydrate on egg laying became stronger as cholesterol was increased to 0. 15 g/l (Figure 2d) and 0. 3 g/l (Figure 2f), with no further change as cholesterol increased from 0. 3 g/l to 0. 6 g/l (Figure 2h). This increasingly negative relationship between carbohydrate and egg laying was caused because increasing cholesterol benefited egg laying more at lower dietary carbohydrate levels – the opposite of what we observed for lifespan. Thus, once again fly lifespan and egg laying worsened as cholesterol was diluted, but unlike its negative interaction with increasing dietary protein, the detrimental effects of lowering cholesterol became stronger as carbohydrate levels decreased. This indicates that the negative impact of lowering cholesterol is not a specific interaction with either high protein or low carbohydrate levels in the diet. We also found that the benefits of cholesterol addition were not related to the caloric content of the diet because cholesterol improved fecundity and extended lifespan of flies on almost all diets, including those with the lowest, intermediate, and highest caloric densities (Figure 2—figure supplement 2). Instead, lowering cholesterol produces worse outcomes as the dietary protein: carbohydrate ratio increases. This is the same change in macronutrient balance that promotes increasing egg laying. We saw that flies produce more eggs in response to increasing dietary protein: carbohydrate ratio and that these positive effects persist even as dietary cholesterol falls to a level that cannot fully support lifespan (less than 0. 3 g/l cholesterol). Thus, the protein: carbohydrate ratio appears to take precedence over dietary sterol levels in the decision to commit to reproduction. If this is the case, we expect to see a positive relationship between the dietary protein: carbohydrate ratio and egg laying across all levels of dietary cholesterol. This is indeed what we found, although the positive relationship was modified by cholesterol level (Figure 3a, c, e, g, Supplementary file 7), starting with a weak positive effect on 0 g/l cholesterol (Figure 3a) and becoming increasingly positive as cholesterol was increased to 0. 15 g/l (Figure 3c) and 0. 3 g/l (Figure 3e). Once again, increasing cholesterol from 0. 3 g/l to 0. 6 g/l promoted no further benefit (Figure 3g). Reproduction can impose a cost on future survival if resources that are essential for somatic maintenance are preferentially committed to making eggs. Since increasing protein: carbohydrate levels drove increasing egg laying, even when the adults were completely deprived of sterols, it is possible that females are committing sterols to egg production at a rate faster than they can replenish it from the diet. If true, mothers on low cholesterol diets would become shorter lived as egg laying increases, but when cholesterol is sufficient, the relationship between egg production and lifespan should become less negative. To test this, we plotted egg laying against lifespan for all replicates across all diets. This showed that egg laying was a significant predictor of lifespan, and that this relationship was modified by dietary cholesterol (Figure 3b, d, f, h, Supplementary file 8). When the data are grouped by dietary cholesterol level (Figure 3), we see that when cholesterol was at 0 g/l (Figure 3b), there was a negative relationship between egg laying and lifespan, but as the cholesterol level increased, the correlation flattened, such that the slope was no longer negative for each level of cholesterol supplementation (Figure 3d, f, h, Supplementary file 8). Thus, when dietary cholesterol was insufficient, increasing dietary protein: carbohydrate drove higher egg laying (Figure 3a) and this predicted lifespan shortening (Figure 3b) – a scenario that exemplifies the negative relationship between reproduction and lifespan in response to increasing protein: carbohydrate levels that is regularly reported in the dietary restriction literature (Mair et al., 2005; Lee et al., 2008; Skorupa et al., 2008; Solon-Biet et al., 2014; Solon-Biet et al., 2015; Simpson et al., 2017). However, when cholesterol was increased, the negative relationship was reduced such that egg laying was either completely independent of lifespan (Figure 3d) or became slightly positively correlated, indicating that the dietary conditions, which promote egg laying, are the same as those that promote longer lifespan (Figure 3f, h). Thus, higher egg laying in response to increasing protein: carbohydrate levels only shortens lifespan when cholesterol is insufficient to support egg production. TOR signalling is a key amino acid signalling pathway that is critical for growth, reproduction, and lifespan. Because TOR activity increases with dietary protein levels, it has been implicated as mediating the detrimental effects on lifespan of high protein diets (Liu and Sabatini, 2020). This is supported by the fact that rapamycin, a pharmacological suppressor of TOR, has been shown to extend lifespan across numerous species, including Drosophila where it also suppresses egg production across different caloric densities (Bjedov et al., 2010; Schinaman et al., 2019; Scialò et al., 2015). If sterol limitation is the reason why high egg production on high protein: carbohydrate diets causes reduced lifespan, rapamycin might extend lifespan because it reduces egg production and therefore rescues females from sterol depletion. If true, rapamycin should extend life only when the flies on high protein: carbohydrate diets are sterol limited. As before, when we maintained flies on a high protein: carbohydrate diet, increasing dietary cholesterol from 0. 1 to 0. 3 g/l increased lifespan (62 d median v 69 d median) (Figure 4a). Egg laying was also slightly (34%), but significantly, elevated by cholesterol supplementation (Figure 4b) indicating that 0. 1 g/l cholesterol was limiting for both lifespan and reproduction. When rapamycin was added to both foods, egg laying was almost completely suppressed (Figure 4b). Rapamycin also extended fly lifespan, but only for flies on low dietary cholesterol (0. 1 g/l) (Figure 4a), bringing their lifespan up to the same level as flies on higher cholesterol food (0. 3 g/l; 69 d median). Adding rapamycin to the food with higher cholesterol did not result in any additional lifespan improvement over what was already achieved by increasing cholesterol alone (69 d median; Figure 4a). These data show that lifespan extension by rapamycin administration is conditional on the flies being on a low cholesterol diet. Together, our data are consistent with the flies’ lifespan being determined by having access to sufficiently high levels of dietary sterols that they have enough left over after reproduction to meet their needs for somatic maintenance. This can be achieved either by enriching the amount of cholesterol in the diet, or by reducing the flies’ expenditure on egg production, which can be achieved by reducing the dietary protein: carbohydrate ratio or by suppressing egg production pharmacologically. The experiments above were all performed using synthetic diets in which our ability to vary the absolute and relative concentrations of protein, carbohydrate, and sterol are limited only by solubility. However, most laboratories maintain fly populations on a diet that consists of yeast and sugar as well as variable numbers of other ingredients (Piper, 2017). Although the relative concentration of each nutrient in yeast is more constrained than on our synthetic diet, systematic studies have shown that the type and commercial source of yeast can have significant effects on overall dietary composition (Lesperance and Broderick, 2020) and the relationship between lifespan and egg laying (Bass et al., 2007). In Bass et al., 2007, the most dramatic lifespan reduction for increasing yeast was found when the fly food was made with a water-soluble extract of yeast that would contain very little, if any, sterols. Thus, similar to what we demonstrated on the synthetic diet, the shortening of fly lifespan when increasing the yeast content (protein: carbohydrate ratio) in lab foods may be caused by an insufficiency of dietary sterols. We tested the effects of supplementing cholesterol into two sugar/yeast recipes that have been commonly used to study the effects of dietary restriction on lifespan (Mair et al., 2005; Bass et al., 2007; Katewa et al., 2016). These diets differ in both the number of ingredients used and the type of yeast; while both are Saccharomyces cerevisiae, one is a whole cell autolysate, while the other is a water-soluble extract. Adding 0. 3 g/l cholesterol to both the low yeast (dietary restriction) and high yeast foods of both yeast types had a significant positive effect on lifespan (Figure 5a, c) and egg laying (Figure 5b, d) when compared to diets without cholesterol supplementation. The magnitude of this benefit to lifespan was greater for flies on the high yeast foods than on the low yeast foods, meaning that cholesterol supplementation narrowed the difference between the dietary restriction vs high yeast diet from 9 to 4% for flies on the autolysed yeast diets (Figure 5a) and from 81 to 25% lifespan extension for flies on the yeast extract diets (Figure 5c). We note that even with cholesterol supplementation, the flies on the high yeast diet were still significantly shorter lived than those on the cholesterol supplemented low yeast food. This small additional cost of the high yeast food could reflect a detrimental (toxic) effect on lifespan of very high dietary protein, similar to what we observed in our highest protein diets on the synthetic foods (Figure 2e, g). This is not rescuable by cholesterol supplementation and is not related to the number of eggs that females produce. In the lab, flies can be successfully reared and maintained on a mixture of just sugar and yeast (Pearl and Parker, 1921). This diet is thought to reflect their natural diet of rotting fruit and the microbial community – principally the yeasts – that cause the fruit to decay (Markow et al., 2015; Piper, 2017). Yeast contains all of the nutrients that flies require, including protein (~45%), carbohydrate (~40%), a small amount of fat (~7%), nucleic acids (~7%), and micronutrients, such as sterols, metal ions and vitamins, which are essential for flies. Drosophila rely heavily on protein from yeast, as well as carbohydrate from both yeast and plant sources, to guide their feeding behaviour. They select amongst foods containing the appropriate protein and carbohydrate concentrations to enhance their fitness (Ribeiro and Dickson, 2010; Vargas et al., 2010; Walker et al., 2017). Many of the other nutrients from their diet, including sterols, do not affect feeding behaviour, presumably because they are normally acquired in adequate quantities as part of a diet that is sufficient in macronutrients (Walker et al., 2015; Leitão-Gonçalves et al., 2017; Münch et al., 2020). While the proportion of protein and carbohydrate in yeast remains relatively constant across growth conditions, the abundance of sterols can vary over a 10-fold range in response to changes in oxygen availability, which is essential for sterol biosynthesis (Starr and Parks, 1962; Wilson and McLeod, 1976). Thus, because fly feeding behaviour and egg production are almost entirely shaped by the macronutrients, fly lifespan is susceptible to reductions in the sterol: protein content of dietary yeast. Our data indicate that this is because protein drives sterols to be preferentially partitioned towards reproduction at the expense of maintaining the adult soma. While we have found this to be the case for flies feeding on lab based foods, it is also reasonable to expect it for flies feeding on rotting fruit, where microbial growth is largely fermentative (driven by high sugar levels and limiting oxygen), producing ethanol and short chain acids to which Drosophila has evolved a healthy tolerance (Geer et al., 1993). There have been several theoretical attempts to describe the mechanistic basis for the lifespan benefits of dietary restriction (Blagosklonny, 2006; Blagosklonny, 2010; Kirkwood and Rose, 1991; Moatt et al., 2020; Regan et al., 2020; Speakman, 2020). In particular, the disposable soma theory proposes that organisms will strategically reallocate nutrients towards somatic maintenance at the cost of reproduction when nutrients are scarce and that this enhances lifespan (Kirkwood and Rose, 1991). Our data indicate that this trade-off can exist for flies feeding on yeast, but only when dietary sterols are limiting. However, when dietary sterols are not limiting, this trade-off does not need to exist and a single nutritional optimum for both lifespan and reproduction can be found. Thus, the macronutrient balance that drives higher egg laying does not necessarily inflict a direct cost on lifespan. In mechanistic work, the increased lifespan under dietary restriction has been attributed to the benefits of reduced dietary protein, which enhances proteome maintenance via reduced TOR signalling (Harrison et al., 2009; Partridge et al., 2011; Kapahi et al., 2017; Piper et al., 2017; Sabatini, 2017; Dobson et al., 2018; Liu and Sabatini, 2020). Interestingly, lysosomal cholesterol levels have recently been found to be a potent modifier of mTORC1 activity (Castellano et al., 2017; Zhang et al., 2020), which raises the possibility that both protein depletion and cholesterol addition modify ageing by reducing TOR signalling. However, the published data shows that cholesterol is an activator of TOR and cholesterol depletion inhibits its activity. Thus, we expect adding cholesterol to the diet would not reduce TOR signalling, but instead optimise conditions for maximal TOR signalling – especially on the high protein: carbohydrate diets in which we find cholesterol addition to be most effective for prolonging lifespan. These data indicate, therefore, that long life is possible when TOR signalling is high as long as the flies have sufficient sterols in their diets. Alternatively, longevity can still be assured under sterol-limiting conditions by reducing the cost of reproduction, either by reducing dietary protein, by adding rapamycin which suppresses reproduction or by making flies infertile (Wu et al., 2020). These data indicate that longevity assurance in D. melanogaster is not the result of enhanced proteostasis triggered by lowered TOR, but is instead, a side effect of avoiding sterol depletion caused by an over-investment in egg production. Rapamycin is known to extend the lifespan of various organisms including C. elegans, yeast and mammals (Harrison et al., 2009; Kapahi et al., 2010; Powers et al., 2006; Robida-Stubbs et al., 2012). Because C. elegans cannot synthesise its own sterols, rapamycin might increase lifespan by preventing sterol depletion in a manner similar to what we have observed in Drosophila. However, sterols may not be lifespan limiting in other organisms such as yeast and mammals that have the ability to synthesise their own sterols. One explanation is that the administration of rapamycin prevents other micronutrient deficiencies caused by over-investment in growth and/or reproduction in response to high levels of dietary protein. For instance, rodents will export calcium from their own bones and teeth to meet the demands of pregnancy and lactation (Miller and Bowman, 2004; Ozbek et al., 2004; Speakman, 2008). For this reason, it would be interesting to see if providing additional micronutrients to the diets of ad libitum-fed mice can mimic the benefits of dietary restriction, similar to what we see for sterol supplementation in flies. Another possibility is that rapamycin rescues animals from the effects of protein toxicity, which can occur at concentrations of protein higher than what we used in this study. In our experiments, we limit the upper range of dietary protein concentrations so as not to exceed those that are beneficial to reproduction. This practice is informed by the desire to study how dietary restriction enhances somatic maintenance to extend life in already healthy individuals, rather than studying the reduced risk of dying that occurs when flies are prevented from over-consuming. To test this, it would be interesting to study the effects of rapamycin addition over a broader range of protein concentrations than what we have used. If true, this has the important implication that rapamycin, and indeed different diet compositions, may prolong animal lifespan by more than one molecular mechanism. These are important implications to explore since the majority of work studying ageing in lab organisms is based on the assumption that the mechanisms are evolutionarily conserved, in the hope that they will benefit humans. Our data show that the detrimental effects of a high protein: carbohydrate diet on lifespan in female Drosophila melanogaster are, to a significant extent, driven by an indirect nutrient trade-off, in which the macronutrients drive maternal sterol depletion by enhancing egg laying. This is a fundamentally different mechanism from the predominant view that reducing protein: carbohydrate balance in diets improves lifespan by a direct action to reduce TOR signalling and enhance proteostasis. Because of our discovery, we show that the shortened lifespan of flies on a high protein: carbohydrate diet can be improved by supplementing their diet with cholesterol, as well as by reducing egg production by lowering the dietary protein: carbohydrate ratio or by administering rapamycin. Further work is now needed to discover the mechanisms through which cholesterol works to modify lifespan in Drosophila melanogaster, and the role of other important micronutrients in healthy ageing across taxa. All experiments were conducted using a wild type Drosophila melanogaster strain called Dahomey (Mair et al., 2005). These flies have been maintained in large numbers with overlapping generations to maintain genetic diversity. Upon removal from their population cages, flies were reared for two generations at a controlled density before use in experiments, to control for possible parental effects. Eggs for age-synchronised flies were collected over 18 hr, and the resulting adult flies emerged during a 12 hr window. They were then allowed to mate for 48 hr before being anaesthetised with CO2, at which point females were separated and allocated into experimental vials. Stocks were maintained and experiments were conducted at 25 °C on a 12 hr: 12 hr light: dark cycle at 65% humidity (Bass et al., 2007). For all lifespan assays, flies were placed into vials (FS32, Pathtech) containing 3 ml of treatment food at a density of ten flies per vial, with ten replicate vials per treatment. Flies were transferred to fresh vials every two to three days at which point deaths and censors were recorded and saved using the software package Dlife (Linford et al., 2013; Piper and Partridge, 2016). Fecundity was measured as the sum of the mean number of eggs laid per female once per week over four weeks (commencing on approximately day 8 of the experiment), except for the sugar yeast (SY) medium experiment, for which egg counts were recorded in weeks one, two and three. These timepoints were selected because measuring reproductive output during the first weeks of egg laying has shown to be representative of life-long fecundity in flies (Chapman and Partridge, 1996). The eggs laid on the food surfaces of all vials were imaged using a web camera mounted on a Zeiss dissecting microscope and eggs were counted both manually and using Quantifly (Waithe et al., 2015). Quantifly was trained using five images for each cholesterol concentration due to variance in food opacity. The same methods for making the holidic medium described above were used to make all diets used in the rapamycin experiment. In this case however 18. 9 g/l protein: 17. 1 g/l carbohydrate were used. Cholesterol was added to the diet at a concentration of either 0. 1 g/l or 0. 3 g/l (cholesterol supplemented) and rapamycin was added to a final concentration in the diet of 10 μM. Diets were either un-supplemented, supplemented with cholesterol, rapamycin, or both. Four sugar/yeast (SY) diets were created using sucrose (Bundaberg Sugar, Melbourne Distributors) and either whole yeast autolysate (MP Biomedicals, LLC, #903312) or yeast extract (Bacto Yeast Extract, #212750). These diets correspond to previously published conditions for high protein (fully fed) and low protein (dietary restriction) conditions (Bass et al., 2007; Katewa et al., 2016; Mair et al., 2005). The high protein diets contained, per litre 50 g sucrose and 200 g autolysed yeast, or 50 g sucrose, 50 g yeast extract plus 86 g of cornmeal (The Full Pantry, Victoria, Australia). The low- protein diets contained, per litre 50 g sucrose and 100 g autolysed yeast or 50 g sucrose, 5 g yeast extract plus 86 g cornmeal. To each of these diets, we added cholesterol (Glentham Life Sciences, GEO100, #100IEZ) at a concentration of either 0 or 0. 3 g/l. Cholesterol was added to all diets as a powder which was mixed in with all other dry ingredients prior to cooking. This gave us a total of four experimental diets per yeast. All statistical analyses were performed using R (version 3. 3. 0, available from http: //www. R-project. org/). One outlier was removed from the data set as the total number of eggs laid for that particular vial was more than two standard deviations from the mean. Omitting this point did not modify the significance of any of the statistical analyses or change any conclusions. For each experimental vial the median lifespan and mean number of eggs laid were obtained prior to analysis. Linear mixed effect models were used to analyse all data obtained using the holidic media. For the analysis of data obtained using the holidic media, a model reduction was performed by stepwise removal of the most complex non-significant term until any further removal significantly reduced the model fit. Log rank tests were used to compare the survival curves in the rapamycin experiment and yeast based dietary experiments. Finally, two-way ANOVAs were used to analyse egg laying results for the yeast based experiments and rapamycin experiment. Plots were made in Graphpad Prism (version 8. 4. 2). | Title: A dietary sterol trade-off determines lifespan responses to dietary restriction in Drosophila melanogaster females Summary: For the past fifteen years, animal studies have consistently shown that a low-protein, high-carbohydrate ('carbs') diet can extend the lifespan of many organisms, but at the cost of the number of offspring an individual can produce. Yet, it is still unclear what the best dietary balance is, and how these effects arise. One potential explanation could be that reproduction damages the body: low levels of proteins would therefore prolong life by lowering the reproductive output. Here, Zanco et al. examined the possibility that protein intake in fruit flies could instead be acting indirectly by changing the levels of a fat-like molecule called cholesterol, which is used to maintain the body and to support reproduction. To test this idea, groups of fruit flies were fed high levels of proteins. This led to increased reproduction rates, in turn depleting the mothers' reserves of cholesterol. Without enough of the molecule in their diet, the insects were less able to maintain their bodies, which reduced their lifespan. When Zanco et al. added cholesterol to a high-protein diet, the flies lived for the normal length of time. Longer lifespan therefore did not require restriction of the diet or any of its components. In fact, the flies that lived the longest ate protein rich diets, and reproduced the most. This study helps to better understand why changes in diet can influence how long an organism lives for, highlighting that the abundance of certain key molecules may be more important than restricting the levels of proteins, carbs or calories actually consumed. | 9,359 | 365 | lay_elife | en |
Summarize: A Manhattan ad executive who went out to smoke on the balcony of her 17th-floor apartment while on a first date plunged to her death early Thursday morning when she sat on the railing and it gave way. Tracie Strahan reports. (Published Thursday, Aug. 1, 2013) A Manhattan ad executive who went out to smoke on the balcony of her 17th-floor apartment while on a first date plunged to her death early Thursday morning when she sat on the railing and it gave way, according to police sources. Sources told NBC 4 New York that Jennifer Rosoff, 35, and her date were on the balcony at about 12:30 a.m. and when she sat on the railing's corner, it broke and she plunged to the ground, hitting scaffolding on the way down. Her date told investigators he heard two pops before the railing gave way. Witnesses reported seeing a screaming man, believed to be Rosoff's date, running from the building at 400 E. 57th St in Sutton Place. Police spoke to him and cleared him of any wrongdoing. Building Inspected After Balcony Gives Way, Woman Falls to Death The I-Team looks into what could have gone wrong on the balcony that gave way in midtown when a woman sat on it and fell to her to death. Andrew Siff reports. (Published Monday, Aug. 5, 2013) A source on the scene provided a description of the railing, which appeared to be metal, and was bent and twisted, with a cigarette lighter still sitting nearby. The Buildings Department is investigating. Only the higher-floor corner apartments in the gray brick building have balconies, and the Buildings Department issued a partial vacate order for them Thursday. A neighbor of Rosoff on the 17th floor, who did not want to give his name, told NBC 4 New York his landlord had sent the superintendent and an inspector to examine his balcony two or three months ago. "They just asked to peek at it, and I just assumed they were only talking about the surface of it, because that's what they had worked on," he said. A mother who also lives in the building said she was worried for her daughter's safety after the tragedy. "I hope they're checking a lot more than the terraces," she said. "Everything should be checked." The landlord, Stonehenge Management, LLC, did not immediately respond to requests for comment. Rosoff, who had worked at The New Yorker and Lucky Magazine, was director of sales at TripleLift, an advertising company on Fifth Avenue. CEO Eric Berry told NBC 4 New York the staff was struggling Thursday after learning of her death. "She was a well-loved and highly respected member of our team," Berry said in a statement. "The entire company is distraught by the loss of Ms. Rosoff -- she will be deeply missed." Samantha Massa, an acquaintance of Rosoff, told NBC 4 New York said "she was ambitious, she had a great career." Rosoff's family said funeral services were scheduled for this weekend. NEW YORK (CBSNewYork/AP) — A 35-year-old woman fell to her death from the 17th floor balcony of her Midtown Manhattan apartment after the railing apparently gave way, police said. Jennifer Rosoff stepped out to smoke a cigarette on the balcony of her corner apartment at a building known as Stonehenge 57 at First Avenue and 57th Street shortly before 1 a.m. Thursday, 1010 WINS’ Al Jones reported. She was sitting on the railing when it suddenly collapsed, causing her to fall backwards, CBS 2’s Don Champion reported. Rosoff, an ad executive, then plunged 17 stories and landed on first-floor construction scaffolding. [cbs-audio url=” http://cbsnewyork.files.wordpress.com/2013/08/alex-balcony.mp3″ ; size=”340px” download=”false” name=”Woman Killed In 17-Story Fall From Midtown Manhattan Balcony” artist=”WCBS 880’s Alex Silverman Reports”] Investigators said Rosoff was on a first date at the time of the incident. Moments before the fall, her date told her she probably shouldn’t lean on the railing and then heard loud snapping sounds before she fell, authorities said. Police spoke to the man and alcohol and foul play have been ruled out as possible causes of the incident. Responding officers found Rosoff unconscious and unresponsive, police said. She was pronounced dead at the scene. Residents of the building were shocked on Thursday. “I’m very sorry to hear what happened,” one resident said. “The building is very well maintained so I was surprised when I heard the announcement.” A neighbor who has been in the apartment in question said the balcony is very narrow. “You’ve got about two feet or less,” neighbor Sara Shubert said. “It’s for standing and smoking and looking at the moon or whatever.” Shubert added she’ll never forget the sound of Rosoff’s body landing on the scaffolding. “I had no idea, but I’d never imagined that it was what it was,” she told WCBS 880’s Alex Silverman. “A dull, big thud.” The city’s Department of Buildings was at the scene Thursday examining the stability of other balcony railings. It issued an order to residents to stay off their balconies until they are all inspected, Jones reported. Only the upper floor apartments have balconies in Rosoff’s building. [cbs-audio url=” http://cbsnewyork.files.wordpress.com/2013/08/balcony-1-jones-w41-jpowers.mp3″ ; size=”340″ download=”false” name=”Woman Killed In 17-Story Fall From Midtown Manhattan Balcony” artist=”1010 WINS’ Al Jones reports”] Rosoff worked at The New Yorker, Lucky Magazine and Cosmopolitan before recently joining a new media advertising startup called TripleLift, according to her LinkedIn profile. In a statement, her boss said coworkers were distraught, saying Rosoff was a “well-loved and highly respected member of our team. Her tremendous energy and humor brought so much joy to the office.” She attended Tulane University in New Orleans. “It shakes you up. It’s a shame,” Shubert said. Stonehenge 57 issued the following statement: “By now most of you have probably heard about the recent tragedy. Our sincere condolences go out to the family and friends of Ms. Rosoff. We have reached out to the family to express these sentiments personally. We are cooperating fully with the investigation into the cause of this terrible accident. If you have any questions please contact Jason Dworkin at 646-381-7799 during normal business hours.” There was no immediate word how long residents in the building will have to stay off their balconies. City inspection reports did not find any recent complaints or violations pertaining to balconies in the building, Champion reported. You May Also Be Interested In These: (TM and © Copyright 2013 CBS Radio Inc. and its relevant subsidiaries. CBS RADIO and EYE Logo TM and Copyright 2013 CBS Broadcasting Inc. Used under license. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed. The Associated Press contributed to this report.) | Summary: An ad executive on a first date fell to her death when the railing on a 17th-floor balcony in midtown Manhattan collapsed, reports NBC New York. Police say 35-year-old Jennifer Rosoff went out for a smoke on her apartment's balcony just after midnight today and disregarded her date's advice not to sit on the railing, reports CBS New York. It quickly gave way, and she was pronounced dead at the scene. Police have ruled out foul play and cleared her suitor. "You've got about two feet or less," says a neighbor of the balcony. "It's for standing and smoking and looking at the moon or whatever." | 1,708 | 152 | multi_news | en |
Summarize: Nearly 100,000 people have signed a petition calling for the Spanish health minister to resign over her order to put down the beloved pet dog of the nurse who beat Ebola. Teresa Romero Ramos survived the deadly virus after she became the first person to contract it outside Africa while treating two infected missionaries. However, while she and her husband were in quarantine, Spanish authorities put down the family dog Excalibur on October 8 - claiming it could have spread the disease. Anger: Nearly 100,000 people have signed a petition calling for the Spanish health minister to resign over her order to put down Exclaibur, the beloved pet dog of nurse Teresa Romero Ramos who survived Ebola. Excalibur's death provoked widespread protest among animal rights groups, with angry demonstrators taking to the streets of Madrid, calling for the resignation of the country's health minister Ana Mato. Now campaign group Partido Animalista, or PACMA, have presented a petition signed by nearly 100,000 people to the Spanish parliament urging her to quit, along with Madrid health officials Javier Rodriguez and Julio Zarco. However, the Spanish government have rejected the petition and she will remain in office. Health Minister: Campaign group Partido Animalista have presented the petition to the Spanish parliament urging Ana Mato (pictured) to quit. Mrs Romero Ramos, 44, who was placed in quarantine on October 6 after her Ebola diagnosis, has described learning of the death of her pet as the worst part of her ordeal. The animal was put to sleep inside Mrs Romero Ramos’s home, which was disinfected before the animal’s body was taken away in a white van to a nearby incinerator. The decision to put down Excalibur, without testing the animal to see whether it was infected, caused widespread protests. Demonstrators who mounted a vigil outside to try to stop the move shouted ‘murderers’ and several threw themselves on the ground as the vehicle left. Some 300,000 people had already signed a petition urging authorities to spare Excalibur. Twitter was awash with photographs of dogs, cats and birds which were posted alongside the hashtag 'SalvemosAExcalibur' – Spanish for 'Let's save Excalibur'. 'Teresa's dog Excalibur was sacrificed despite the opposition of her owners, thousands of citizens and experts, without evening knowing if it has been infected (with Ebola) and knowing that there are no studies which show that Ebola can be transmitted from dogs to humans,' Pacma said in a statement, according to news website The Local. Many people in Spain felt that the dog's demise was a reflection of how the country's authorities handled Mrs Romero Ramos's disease, with a string of mistakes by health co-ordinators. Mrs Romero Ramos caught Ebola after treating Spanish missionary Manuel Garcia Viejo, 69, who died on September 25 after being infected with the disease while he treated patients in Sierra Leone. She has admitted touching her face with her gloves as she took off a protective suit after leaving the room of one of the priests. Demonstration: The decision to put down Excalibur, without testing the animal to see whether it was infected, caused widespread protests. Protest: Jose Ramon (centre, in black), Mrs Romero Ramos's brother holds a banner along with other protesters that reads 'We demand authorities to respect profesionals. All with Teresa' in Madrid. Ms Mato initially faced calls for her resignation after it emerged that Mrs Romero Ramos had complained of feeling unwell six days before she was eventually admitted to hospital. She was rushed to hospital by unprotected paramedics in a normal ambulance only taken out of service 12 hours later and found out she had Ebola by reading a Spanish newspaper website as she waited to be quarantined. Her home in Alcorcon, near Madrid, that she shares with husband Javier Limon Romero who was also quarantined, was not disinfected until October 8, two days after her diagnosis. Another 13 people who had come into contact with her for 21 days - the incubation period for the disease - though all have now been cleared. Recovery: Mrs Romero Ramos was treated with a drip of human serum containing antibodies from Ebola sufferers who had survived the disease. Query: At a news conference last week, her husband Javier Limon Romero questioned why the decision had been taken, saying the animal had not been to blame 'for anything' Mrs Romero Ramos was treated with a drip of human serum containing antibodies from Ebola sufferers who had survived the disease, and other drugs which a government spokesman declined to name. Mrs Romero Ramis said: 'The worst part was in regard to my dog. Now I'm going to get a puppy. A little Excalibur.' Mrs Romero has tested negative for the infection twice in the last week, with 48 hours between each test, but will remain in hospital in Madrid as a precaution until she is fully recovered. Speaking about her recovery, she added: 'I feel newborn, that is how I feel. I have had a very hard time. I told [my family] I was feeling very bad. 'I told them that I was sick, that I was feeling awful. But I also told them to stay calm. 'What they do is to cheer me up. They tell me they are longing to take off their protective suits to hug and kiss me. 'They cheer me up constantly and ask my what I need all the time. I am thankful to death for them.' At a news conference last week, her husband questioned why the decision had been taken, saying the animal had not been to blame 'for anything'. Earlier this week he described the animal as 'the son we never had', and adding that authorities had given him no chance to argue against the decision. In the U.S. a dog belonging to a nurse infected with Ebola was quarantined and tested negative, before being released | Summary: Teresa Romero Ramos contracted virus while treating infected missionaries. While she was in quarantine Spanish authorities put down her dog Excalibur. Health officials did not test the animal, but said it could have spread disease. Death sparked calls for the resignation of health minister Ana Mato. Animal rights group PACMA have submitted petition calling on her to quit. Mrs Romero Ramos says Excalibur's death was the worst part of her ordeal. She is recovering from the disease but is still in hospital as a precaution. | 1,336 | 112 | cnn_dailymail | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATION This is a continuation of 09/416,661 filed on Oct. 12, 1999 now U.S. Pat. No. 6,309,350 which is a continuation-in-part of application Ser. No. 09/303,634, filed May 3, 1999 now abandoned. BACKGROUND OF THE INVENTION I. Field of the Invention This invention relates generally to medical apparatus for monitoring physiologic parameters within the body of a human or other animal, and more particularly to an implantable device for chronic monitoring of pressure, flow and temperature within living humans or animals. II. Discussion of the Prior Art In the diagnosis and treatment of various maladies, a variety of devices have been developed which can be implanted within the body and used to monitor various physiologic parameters. With the advent of microminiature circuitry, it has become practical to implant a variety of sensors responsive to various physiologic changes, along with circuitry for the transcutaneous transmission of information from the implanted unit, via a telemetry link to an external recording/display device. For example, in the field of implantable cardiac pacemakers and defibrillators, sensing circuitry is incorporated therein for monitoring a number of physiologic parameters, such as respiratory rate, tidal volume, heart rate, blood temperature, movement, etc. Pacemaker leads have been developed that incorporate pressure transducers and temperature sensors such that the pacing rate of the implanted device can be made to vary in relation to detected changes in blood temperature and blood pressure. In implementing such devices, the electronic circuitry is housed in a body compatible, fluid impervious housing along with a suitable power supply or AC to DC converter and electrical leads are then routed from the implant site and through the vascular system to a location on or in the heart. Because of concern that the presence of a lead in the left ventricular chamber may result in the formation of a thrombus that could break loose and reach the brain and cause stroke or embolize to another peripheral vessel, pacing leads or other devices are seldom inserted into the left ventricle, especially for chronic monitoring or therapy delivery. The ability to measure left ventricular pressure or its surrogate in the ambulatory patient, non-invasively, has great potential in determining the status of heart failure patients, providing an opportunity to modify medical management of ventricular dysfunction very precisely as compared to current clinical practice. Moreover, ambulatory hypertensive patients can be managed more closely when peak systolic and diastolic pressure can be chronically monitored. The ability to measure myocardial temperature with an implanted device and to thereafter telemeter the temperature information to an external monitor will permit cardiac transplant patients to be closely managed. It is believed that rejection in organ transplant patients manifest early as a small tissue temperature elevation due to the inflammatory reaction of rejection. The only presently available method to determine transplant status is to perform a biopsy, an invasive procedure that is sometimes done weekly or more often, and is done in such a patient hundreds of times during that patient's life. A device for measuring tissue temperature and telemetering the information to an external monitor would limit the number of times such biopsy is required—a significant clinical advance. Myocardial temperature sensing is beneficial in the management of heart failure. Ventriculo-vascular coupling and impedance mismatches manifest themselves as excess heating of the ventricle. By having temperature monitoring available, accurate titration of preload and afterload reducing medication could be achieved to limit myocardial energy output and thereby the heart will perform more efficiently. Therefore, a need exists for a system for chronically monitoring temperature and pressure within the left ventricular and/or atrial chambers of the heart or myocardial tissue. It has also determined that a temperature sensor located in the pulmonary artery branches for sensing lung tissue temperature can provide meaningful information following heart/lung transplant surgery in that an elevated blood or lung tissue temperature in the pulmonary artery or branches may be indicative of the onset of rejection, allowing interventional adjustment in the amount of anti-rejection drug being administered to, the patient. We are presently unaware of any temperature sensor that can be chronically implanted to measure temperature changes in blood traversing the pulmonary artery. By locating the monitor implant at other locations within the body, renal, hepatic or pancreas transplant status can be assessed. Locating the device in the peripheral blood vessels can allow assessment of exercise capacity. The monitor may also be used to calculate blood flow using thermodilution techniques. From the foregoing, it can be seen a need exists for an implantable sensor especially designed for placement in a selected portion of a patient's vascular system and which can be used to chronically transmit pressure and/or temperature data to an external monitor/display unit so that a medical professional can more readily diagnosis and treat various medical conditions. It is principal object of the present invention to fulfill this need. SUMMARY OF THE INVENTION In accordance with the present invention there is provided a medical monitoring apparatus that comprises a support member that is adapted for chronic implantation at a predetermined location within the vascular system of a living human or other animal. One or more sensor devices are affixed to the support means for sensing at least one measurable physiologic parameter. The apparatus further includes a means for telemetrically transmitting signals representative of the sensed parameter percutaneously to an external signal receiver. In accordance with one embodiment of the invention, the support means may comprise a self-expanding or balloon expandable tubular stent that is adapted for chronic implantation at a predetermined location in the vascular system and affixed to the tubular stent is an electronic circuit for measuring a physiologic parameter. The electronic circuit means also includes a means for telemetrically transmitting signals representative of the sensed parameter percutaneously to a signal receiver external to the body of the living animal. To measure left ventricular pressure/temperature, the apparatus of the present invention may be placed in an puncture made through the ventricular septum with the stent being anchored in this opening, such that the pressure/temperature sensor is exposed to blood or tissue in the left ventricle. An anchoring arrangement is provided on the stent to prevent the normal pumping action of the heart from displacing the implanted stent. To prevent blood flow through the tubular stent, the lumen thereof may be packed with a fibrous material for occluding the opening. The electronics module may also be located in the lumen if occlusion is desired. If the stent device is to be placed in the pulmonary or some other artery of a patient, the anchoring means may comprise a series of hooks that become engaged with the inner wall of the artery when the stent is allowed to or made to expand radially during its implantation. DESCRIPTION OF THE DRAWINGS The foregoing features, objects and advantages of the invention will become apparent to those skilled in the art from the following detailed description of a preferred embodiment, especially when considered in conjunction with the accompanying drawings in which like numerals in the several views refer to corresponding parts. FIG. 1 is a perspective view showing a tubular stent as a support member for an electronic circuit package for sensing and telemetrically transmitting sensed pressure and temperature data and powered by an implantable power pack; FIG. 2 is an end view of the device of FIG. 1; FIG. 3 is a sectioned view through the heart showing the monitor device of the present invention located in the ventricular and atrial septum; FIG. 4 is a schematic diagram illustrating apparatus for applying power to an implanted unit percutaneously; and FIG. 5 is a block diagram of the integrated circuit chip forming a part of the implantable monitor apparatus. DESCRIPTION OF THE PREFERRED EMBODIMENT Referring first to FIG. 1, there is illustrated a first embodiment of a temperature/pressure monitoring device adapted for placement at a desired location within the vascular system of a living animal. It is seen to comprise a support member 10, here shown as a self-expandable or balloon expandable stent, to which is attached an electronics module 12 that is adapted to be powered by an implantable power source 14 connected to it by means of conductors 16. The power source 14 is preferably a lithium-iodide battery contained within a body fluid impervious housing 18. The electronic circuitry comprising the module 12 is also contained within a body fluid impervious housing 20 having sealed electrical feed-throughs 22 to which the conductors 16 are attached for bringing DC power into the module. As will be further explained, associated with the electronics module 20 are one or more sensors for detecting changes in a physiologic parameter such as blood temperature, blood pressure or flow. The sensor may comprise a pressure sensor of the type described in the Brockway et al. U.S. Pat. No. 4,846,191, either alone or in combination with a thermistor temperature transducer and a Doppler flow sensor. Formed on opposed ends of the stent 10 are retention elements, shown in FIG. 2 as hooks 26 which are adapted to engage tissue to prevent migration of the device from its desired implant site. The need for retention elements is, of course, somewhat dependent on the location selected for the implant. The sectional view taken through a heart illustrated in FIG. 3 shows the way in which the present invention can be used to monitor either left ventricular pressure or left atrial pressure on a chronic basis. Here, an incision is made through the ventricular septum 28 or the atrial septum 30 with a device like that shown in FIG. 1 percutaneously implanted via an artery or vein and inserted into the surgically created opening. The support device 10, itself, may comprise a septal defect occluder fashioned after that described in the Kotula et al. U.S. Pat. No. 5,725,552 but with an electronics module 12 mounted thereon. The sensor element is exposed to the blood in the left ventricle and/or the left atrial chamber depending on the placement of the device. The support device 10 may be delivered by way of a catheter routed through the vascular system into the right ventricle and thence through the surgically created septal opening. When the device 10 is released from the confines of the catheter, it self-expands to a predetermined dumbbell configuration, as illustrated, to maintain it in position in the septal wall. Alternatively, in an open heart surgery, the device of FIG. 1 can be inserted through the myocardium of the left ventricle or left atrium. When disposed in the lumen of a blood vessel, the support device 10 is tubular as shown in FIG. 1, permitting blood flow therethrough. The hooks 26 on opposed ends thereof serve to anchor the device in place in the selected blood vessel. Placement of the stent with its temperature/pressure/flow measuring circuitry in the pulmonary artery or a branch thereof can be used to obtain a good estimation of left ventricular end diastolic pressure which is meaningful in the treatment of CHF and hypertension. It is calibrated by direct comparison with left ventricular pressure measured with an acutely placed pressure sensing catheter. Periodic recalibration can be accomplished via software. FIG. 4 illustrates an alternative embodiment of the invention wherein the implant device may receive its operating power transcutaneously from a programmer transducing head 32 supported on a shoulder strap 34 which keeps the transducing head is oriented in alignment with the implanted device. The transducing head 32 may be of the type used in the telemetry link of a programmable implantable pacemaker allowing the patient to be ambulatory. The transmitting and receiving electronics and the battery power supply therefore may be contained in a case 36 worn on a belt surrounding the patient's abdomen. Information developed by the sensor 24 of the implant device 10 is telemetered to the external transducer 32 via RF transmission and is fed to the electronic module 36 for signal processing, storage and later analysis. FIG. 5 is a block diagram illustrating the circuitry contained within the housing 20 of the implant device. The output signals from the aforementioned pressure/temperature/flow transducers can readily be separated into two channels, one for carrying the pressure information and the other for carrying temperature information by appropriate filtering techniques, it being recognized that the output signal from the pressure sensor will be of a significantly greater frequency than that from the temperature sensor. Hence, in FIG. 5, both a pressure sensor 50 and a temperature sensor 52 are illustrated to indicate the dual channel nature, even though a single transducer device may be utilized. The analog output signal from both the pressure sensor 50 and the temperature sensor 52 are applied to an analog-to-digital converter forming a part of the on-board microprocessor 54. The microprocessor 54 includes an address bus 56, a data bus 58 and a control bus 60 to which are connected a ROM memory 62, a RAM memory 64 and an input/output interface 66. ROM 62 conventionally stores a program executable by the microprocessor 54 while RAM 64 may store programmable constants and intermediate data developed during the execution of the program. The I/O interface is attached to a telemetry circuit 68, allowing data carried on the data line 58 from the microprocessor and/or the RAM to be transmitted transcutaneously from the patient's body, represented by dashed-line 70 to an external monitor 72. The monitor 72 may be conveniently be a lap-top PC having the ability to receive and process the telemetry data from the implant and to deliver programming data to the implant device, via the telemetry link. The temperature transducers illustrated in FIGS. 1 and 2 may comprise a thermistor, or thermocouple or an infrared sensor. A separate piezoelectric device can be utilized as a pressure sensor in a fashion indicated in the Brockway U.S. Pat. No. 4,846,191. It is also contemplated that a separate flow sensor may be made a component of the implantable monitor device or, alternatively, the temperature sensor may be used to assess flow using known thermodilution techniques. This invention has been described herein in considerable detail in order to comply with the patent statutes and to provide those skilled in the art with the information needed to apply the novel principles and to construct and use such specialized components as are required. However, it is to be understood that the invention can be carried out by specifically different equipment and devices, and that various modifications, both as to the equipment and operating procedures, can be accomplished without departing from the scope of the invention itself. | Summary: A medical monitoring apparatus designed to be implanted in the vascular system is capable of sensing and transmitting via a telemetry link to an external monitor both pressure and temperature information. An internally or externally powered microcircuit component is supported on a stent-like structure and adapted to be placed in the vascular system. Placement in the ventricular septum permits measurement of pressure and temperature in the left ventricle without introducing thrombus generating materials in the left ventricle. | 3,335 | 104 | big_patent | en |
Summarize: Updated with Megyn Kelly tweets: Playing out in the press and social media as much as the conference room, it looks like negotiations over Megyn Kelly’s departure from NBC News have taken a bad turn yet again – and the ex-morning show host’s attorney wants the big boys to step in if news boss Andy Lack can’t or won’t stop the leaks. At the same time, Kelly herself is accusing certain outlets of crossing the privacy line in regards to her family. “Despite my efforts to handle this process confidentially, NBC News is allowing the media to run with completely false and irresponsible reports that disparage Megyn by erroneously claiming she has ever asked for more money than her contract requires,” lawyer Bryan Freedman said today after it popped up in the Daily Mail and other outlets that Kelly was now looking for an extra $10 million over the remainder of her estimated $69 million three-year contract with the Comcast-owned company. In talks with NBC News as recently as Tuesday, the LA-based attorney then turns his spotlight on the often porous Lack-run unit for spreading its own narrative and seemingly sitting on its hands simultaneously “If NBC News is not the source then they have a responsibility as a news division to correct these false claims,” Freedman adds. “Or are they somehow attempting to use these fabrications for some fictitious advantage in the discussions we’re having?” Then, from a man who floated getting ex-NBCer Ronan Farrow to sit in on the talks, there’s the kicker squarely aimed to resonant in the corporate corridors and up the chain of command: “If Andy Lack has lost control, my hope would be that Steve Burke can step in and not permit blatant lies about our discussions to remain uncorrected.” Issues of Kelly’s payout, non-compete agreement and the signing of a NDA have been reported by various outlets including Deadline over the last week since Megyn Kelly Today was formally canned on October 26, just days after the host made deeply offensive comments about “blackface” Halloween costumes Today NBC News today didn’t give much credence to the scathing accusations or shout-out to big boss Burke by Kelly’s high-profile lawyer. “Unlike Mr. Freedman, who has repeatedly commented to the media throughout the negotiations, we respect the confidentiality of the process, and will have no comment until it reaches its conclusion.” a NBC News spokesperson told Deadline. Quiet for the most part over the past week, Kelly herself took to social media this morning to name manes and slam the always upclose and personal Daily Mail for invading the privacy of her family: 1 of 2: For a week paparazzi has been lurking outside my home day & nite. Finally today I took my kids to school. I went out alone 1st, offered them donuts &begged them to just take their pic of me & to leave my kids alone when they emerged. All were nice. Except the Dailymail… — Megyn Kelly (@megynkelly) October 31, 2018 2 of 2: The DailyMail 1st published photos of my husb IN OUR HOME & then *did* photog my kids, trailed us to my daughter’s school, & secretly videotaped my 7-yr-old child (her classmates too) & posted it. THIS IS NOT RIGHT. — Megyn Kelly (@megynkelly) October 31, 2018 It is so on and so not over. Happy Halloween. 2 of 2: The DailyMail 1st published photos of my husb IN OUR HOME & then *did* photog my kids, trailed us to my daughter’s school, & secretly videotaped my 7-yr-old child (her classmates too) & posted it. THIS IS NOT RIGHT. 1 of 2: For a week paparazzi has been lurking outside my home day & nite. Finally today I took my kids to school. I went out alone 1st, offered them donuts &begged them to just take their pic of me & to leave my kids alone when they emerged. All were nice. Except the Dailymail... Kelly's 9 a.m. show was canceled last week, but her contract goes through 2020. So now there's a protracted negotiation taking place -- and in an unusual move, Kelly's lawyer Bryan Freedman is talking about it publicly. "Andy Lack needs to stop," Freedman said Tuesday, accusing the NBC News chair of leaking information about the negotiations. On Wednesday, Freedman upped the ante with another statement. "Despite my efforts to handle this process confidentially, NBC News is allowing the media to run with completely false and irresponsible reports that disparage Megyn by erroneously claiming she has ever asked for more money than her contract requires," Freedman said. "If NBC News is not the source then they have a responsibility as a news division to correct these false claims. Or are they somehow attempting to use these fabrications for some fictitious advantage in the discussions we're having?" Freedman also called out NBCUniversal CEO Steve Burke: "If Andy Lack has lost control, my hope would be that Steve Burke can step in and not permit blatant lies about our discussions to remain uncorrected." NBC fired back with its own statement a few minutes later saying, "Unlike Mr. Freedman, who has repeatedly commented to the media throughout the negotiations, we respect the confidentiality of the process, and will have no comment until it reaches its conclusion." In recent days, several stories from outlets like The Hollywood Reporter and Deadline have said that Kelly and NBC are at odds regarding the amount of money she'll be paid on the way out the door, and regarding the terms of her exit agreement. NBC is said to be reluctant to pay out the rest of her three-year contract, which is reportedly worth $69 million. And Kelly is said to be reluctant to sign away her rights to speak freely. Sources confirmed to CNN that one of the sticking points involves something called a non-disparagement clause, which would prohibit Kelly from speaking ill of NBC in the future. A non-disparagement clause is often a part of a broader non-disclosure agreement, or NDA for short. These types of agreements are common when millions of dollars are on the line. Ann Curry recently confirmed that she signed an NDA when she was forced out of the "Today" show in 2012. Kelly is different because she has repeatedly challenged NBC News management in public, and has hinted that she knows more than she's shared about various NBC controversies. Those cases include the Matt Lauer scandal -- when the host was fired amid misconduct allegations -- and the circumstances surrounding Ronan Farrow's departure from NBC when Farrow was investigating Harvey Weinstein. Kelly's insider knowledge may have given her some leverage in the negotiations. She is reluctant to sign a one-sided NDA, a source said, because she is concerned that would leave her vulnerable to disparagement by NBC executives. But Freedman spoke out earlier when the Daily Mail, citing a source, claimed that Kelly wants an extra $10 million bonus, perhaps in exchange for her silence. Freedman fired back: "Out of respect for the discussions, I am not going to share more details but any suggestion that Megyn is asking or looking for more than her contract is untrue." Freedman said this bonus idea was "clearly planted by NBC News to continue its mission to harm Megyn and gain some sort of leverage. It won't work. Andy Lack needs to stop." In response, Page Six quoted an NBC "insider" saying, "These are desperate attempts to distract from the main issue: Her offensive comments on live TV about blackface." Most of the drama is happening off the air, but NBC News has openly covered the Kelly controversy with multiple segments on television. And Kelly has become a late-night punchline. "Today was national color day," Seth Meyers said on "Late Night" Tuesday night. His writer Amber Ruffin added the punchline: "'So it's okay today, right?' said Megyn Kelly about blackface." Meyers: "So Amber, what are you dressing as for Halloween?" "The ghost of Megyn Kelly's career," Ruffin said. "Oooh, scary." | Summary: Megyn Kelly would like the paparazzi, and one tabloid specifically, to please leave her family alone in the wake of NBC News cancelling her show. On Wednesday she tweeted that "for a week paparazzi has been lurking outside my home day & nite. Finally today I took my kids to school. I went out alone 1st, offered them donuts &begged them to just take their pic of me & to leave my kids alone when they emerged. All were nice. Except the Dailymail..." In the next tweet, she continued, "The DailyMail 1st published photos of my husb IN OUR HOME & then *did* photog my kids, trailed us to my daughter's school, & secretly videotaped my 7-yr-old child (her classmates too) & posted it. THIS IS NOT RIGHT." She wasn't the only one in her camp who referenced the Daily Mail Wednesday: Deadline reports her lawyer, Bryan Freedman, swung at NBC News for "allowing the media to run with completely false and irresponsible reports that disparage Megyn by erroneously claiming she has ever asked for more money than her contract requires." He specifically cited a Daily Mail report that she wants the rest of her $69 million contract (which goes through 2020) plus $10 million as part of her exit package. Freedman said NBC News chief Andy Lack needs to correct the false information and stop leaking it, should the network itself be the source of details of the negotiations. CNN framed Freedman's public comments as an "unusual move," and reports NBC simply replied that it "respect[s] the confidentiality of the process." | 1,901 | 377 | multi_news | en |
Summarize: BACKGROUND OF THE INVENTION The present invention generally relates to medical cardiac pacemakers and more specifically relates to a system and method for optimizing activity threshold in activity based rate adaptive cardiac pacemakers of the type which respond to the patient's metabolic demand and varies the pacing rate in accordance therewith. Early cardiac pacemakers were asynchronous in operation, providing stimulating pulses to the heart at a fixed rate independent of the physiologic demand of the patient. In recent years, pacemakers which measure the metabolic demand for oxygen and vary the pacing rate in response thereto have become widely available. One modern method employed for measuring the need for oxygenated blood includes measurement of patient physical activity by means of an appropriate sensor. Generally, a rate responsive pacemaker which is responsive to patient physical activity includes a sensor which produces an output that varies between a maximum sensor output level and a minimum sensor output level and provides for a pacing rate which typically varies between a selectable lower pacing rate and an upper pacing rate. Such a pacemaker which utilizes a piezoelectric transducer, is disclosed in U.S. Pat. No. 4,485,813, issued to Anderson, et al., and assigned to Medtronic, Inc. It has become common practice in recent years to provide programmable parameters in order to permit the physician to select and adjust the desired parameters to match or optimize the pacing system to the heart patient's physiologic requirements in an effort to minimize patient problems and to prolong or extend the useful life of an implanted pacemaker. Such systems are based upon utilizing a sensor derived variable that is an indicator of the patient's true metabolic and physiologic needs. Activity based rate adaptive pacemakers typically have a programmable activity threshold parameter for adjusting the sensitivity of the circuitry to signals from the activity sensor. Historically, activity threshold parameters in activity based rate adpative pacemakers have been manually programmed and adjusted or optimized in an ad hoc iterative process. Often, because the process is difficult and lengthy, such parameters are not optimized, but left at nominal shipping values. Physicians using activity based rate adaptive pacemakers may have difficulty setting the activity threshold to an optimal setting since this parameter is device specific and there is a marked proliferation of activity based rate adaptive pacemakers by both Medtronic and other companies. Such a parameter will always tend to be device specific more than patient specific, so the physician has no inherent expertise in setting it optimally. An activity based rate adaptive pacemaker which is capable of automatically and continuously determining and adjusting the activity threshold to an optimal setting would both significantly reduce programming time commitment by the physician and insure that activity threshold optimized at any single point in time will continue to remain optimal. One method for dealing with setting the activity threshold to an optimal setting for a given patient is disclosed in the auto set up idea disclosures included in U.S. patent application Ser. No. 07/567,372 entitled Rate Responsive Pacemaker and Method for Automatically Initializing the Same, filed in the name of Roline, et al., on Aug. 14, 1990, and assigned to Medtronic, Inc. The auto set up feature disclosed by Roline is desirable, but has limitations. It requires significant programming timing commitment by the physician. Also, it is unlikely that activity threshold optimized at any single point in time will remain optimal. The location of the pacemaker (subtle movements in the pocket), the IPG pocket characteristics, the patient's tissue composition (gaining/losing weight), the types of patient activities, and vibrational environmental factors may all impact how the activity threshold should be set. While Roline et al. constitutes an improvement over the conventional methods, it has not proven to be completely satisfactory in addressing and resolving the optimization problems associated with the optimization process. It is an object of this invention to overcome the disadvantages and drawbacks of the prior art and to provide a method of activity-based rate-adaptive pacemaking which significantly reduces programming time commitment by the physician or repeated patient sessions in determining and adjusting the activity threshold to an optimal setting, and in which practice the activity threshold optimized at any single point in time will continue to remain optimal. It is a further object of this invention to provide a system for practicing the above said method. SUMMARY OF THE INVENTION The present invention provides a method and system for continually and automatically optimizing and adjusting an activity threshold parameter which is directed to the problem of insuring that the activity threshold optimized at any single point in time will continue to remain optimal without further programming time commitment by a physician or repeated patient sessions. In the present invention, the pacemaker system includes a sensor implantable pacemaker and an external programmer. The system also includes means for continuously optimizing and automatically adjusting the activity threshold to its optimal value. In the preferred embodiment, the process of automatically and continuously optimizing and readjusting an activity threshold parameter includes the steps of setting the activity threshold to an initial value and monitoring the average amount of time at rest in which there is an absence of activity counts. An activity count corresponds to an electrical event which exceeds the activity threshold of the activity sensor. Thereafter, the activity threshold is automatically adjusted to a lower value if the amount of time spent at zero counts is greater than the average amount of time spent at zero counts by more than a predetermined maximum difference value; otherwise, the activity threshold is automatically adjusted to a higher value if the amount of time spent at zero counts is less than the average amount of time spent at zero counts by more than a predetermined minimum difference value. In the preferred embodiment, the readjustment of activity threshold to a new optimized value would occur automatically every 24 hours. However, any time cycle may be used. In another preferred embodiment, the activity threshold is automatically adjusted to a lower value if the amount of time spent at zero activity counts is greater than a predetermined maximum time value; otherwise, the activity threshold is automatically adjusted to a higher value if the amount of time spent at zero activity counts is less than a predetermined minimum time value. BRIEF DESCRIPTION OF THE DRAWINGS The above and other options, features and advantages of the present invention will become more apparent from the following more particular description thereof, presented in conjunction with accompanying drawings, wherein: FIG. 1 is an illustration depicting how a pacemaker system in accordance with the present invention may be implanted in a patient; FIG. 2 is a block diagram representation of the circuitry of a pacemaker system to accomplish automatic activity threshold optimization adjustments according to the present invention; FIG. 3 is a block diagram representation of an activity sensing circuit to process activity sensor signals according to the present invention; FIG. 4 is a simplified flowchart showing the basic function of the software of the pacemaker of FIG. 1 for continuously optimizing and automatically adjusting activity threshold using continuous updates of long term running average of zero activity time for purposes of deriving an optimized pacing rate; FIG. 5 is a simplified flowchart showing the basic function of the software of the pacemaker of FIG. 1 for continuously optimizing and automatically adjusting activity threshold using fixed targets for purposes of deriving an optimized pacing rate; and FIG. 6 is a simplified flowchart showing the basic function of the software of the pacemaker of FIG. 1 for continuously optimizing and automatically adjusting activity threshold using periodic updates of long term running average of zero activity time for purposes of deriving an optimized pacing rate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS FIG. 1 shows generally how a pacemaker 10 in accordance with the present invention may be implanted in a patient 20. A pacemaker lead 30 is electrically coupled to pacemaker 10 and extends into the patient's heart 40 via a vein 50. The distal end of lead 30 includes one or more exposed conductive electrodes 110 for receiving electrical pacing stimuli to the patient's heart 40. Turning to FIG. 2, a block diagram of pacemaker 10 from FIG. 1 is shown. Although the present invention is described in conjunction with a pacemaker 10 having a microprocessor--based architecture, it will be understood that it could be implemented in any logic based, custom integrated circuit architecture, if desired. It will also be understood that the present invention may be utilized in conjunction with other implantable medical devices, such as cardioverters, defibrillators, neural stimulators, cardiac assist systems and the like. In the embodiment shown in FIG. 1, pacemaker 10 includes an activity sensor 100, which may be, for example, a piezoelectric element bonded to the inside of the pacemaker's housing. Sensor 100 provides a sensor output which varies as a function of a measured parameter that relates to the metabolic requirements of patient 20. Pacemaker 10 is schematically shown in FIG. 2 to be electrically coupled via a pacing lead 30 to a patient's heart 40. Lead 30 may be a transvenous endocardial electrode as shown or epicardial electrode (not shown), and can carry either unipolar or bipolar electrodes as is well known in the art. In the preferred embodiment, lead 30 includes an intracardiac electrode 110 located near its distal end and positioned within the right ventricular (RV) chamber of heart 40. In alternative embodiments, intracardiac electrode 110 may be positioned at other locations within the heart such as the left ventricular chamber or the atrium or both as in a dual chamber pacemaker. Electrode 110 is coupled through input capacitor 120 to node 130 and to input terminals of an input/output circuit 140. Input/output circuit 140 contains the analog circuits for interface to the heart 40 and activity sensor 100, as well as for the application of stimulating pulses to heart 40 to control its rate as a function thereof under control of the software-implemented algorithms in a microcomputer circuit 150. Microcomputer circuit 150 comprises an on-board circuit 160 and an off-board circuit 170. On-board circuit 160 includes a microprocessor 162, a system clock circuit 164, and on-board RAM 166 and ROM 168. Off-board circuit 170 includes an off-board RAM/ROM unit 172. Microcomputer circuit 150 is coupled by data communication bus 180 to a digital controller/timer circuit 190. Microcomputer circuit 150 may be fabricated of custom integrated circuit devices augmented by standard RAM/ROM components. It will be understood that the electrical components represented in FIG. 2 are powered by an appropriate implantable battery power source, not shown, in accordance with common practice in the art. An antenna 200 is connected to input/output circuit 140 for purposes of uplink/downlink telemetry through RF transmitter/receiver (RF TX/RX) unit 210. A crystal oscillator circuit 220 provides main timing clock signals to digital controller/timer circuit 190. A Vref/Bias circuit 230 generates stable voltage reference and bias currents for the analog circuits of input/output circuit 140. An analog-to-digital converter/multiplexor (ADC/MUX) unit 240 digitizes analog signals and voltages to provide "real-time" telemetry of intra cardiac signals and battery end-of-life (EOL) replacement function. A power-on-reset (POR) circuit 250 functions as a means to reset circuitry and related functions to a default condition upon detection of a low battery condition, which will occur for example upon initial device power-up or transiently in the presence of electromagnetic interference. The operating commands for controlling the timing of pacemaker 10 are coupled by bus 180 to digital controller/timer circuit 190 wherein digital timers and counters are employed to establish the overall escape interval of the pacemaker, as well as various refractory, blanking and other timing windows for controlling the operation of the peripheral components within input/output circuit 140. Digital controller/timer circuit 190 is coupled to a sense amplifier 300 and an electrogram amplifier 260 for receiving amplified and processed signals picked up from electrode 110 through lead conductor 30 and capacitor 120 representative of the electrical activity of the patient's heart 40. Sense amplifier 300 amplifies sensed electrical cardiac signals and provides this amplified signal to peak sense and threshold measurement circuitry 270, which provides an indication of peak sensed voltages and the measured sense amplifier threshold voltage on multiple conductor signal path 280 to digital controller/timer circuit 190. The amplified sense amplifier signal is also provided to a comparator 290. The electrogram signal developed by EGM amplifier 260 is used in those occasions when the implanted device is being interrogated by an external programmer, not shown, in order to transmit by uplink telemetry a representation of the analog electrogram of the patient's electrical heart activity as described in U.S. Pat. No. 4,556,063, issued to Thompson et al., assigned to the assignee of the present invention and incorporated herein by reference. An output pulse generator 310 provides the pacing stimulus to the patient's heart 40 through coupling capacitor 320 in response to a pacing trigger signal developed by digital controller/timer circuit 190 each time the escape interval times out, or an externally transmitted pacing command has been received, or in response to other stored commands as is well known in the prior art. With continued reference to FIG. 2, input/output circuit 140 further includes sensitivity control circuitry 330 coupled between digital controller/timer circuit 190 and sense amplifier circuit 300. Sensitivity control circuit 330 controls the sense amplifier gain and thus the sensing threshold of sense amplifier 300 as instructed by digital controller/timer circuit 190. Digital controller/timer circuit 190 provides the appropriate information to microcomputer circuit 150 which determines whether the sensitivity threshold of sense amplifier 300 needs to be increased or decreased based on an analysis of sensed electrical cardiac signals. Digital controller/timer circuit 190 is coupled to an activity circuit 400 for receiving, processing and amplifying signals received from activity sensor 100. Activity circuit 400 produces an activity signal which is representative of the patient's metabolic requirements and perpetually maintains the sensing threshold at which patient activity "counts" are recognized at an optimal level in accordance with the present invention. This activity threshold shall be hereinafter described in greater detail with reference to FIGS. 3, 4 and 5. As discussed above, the sensor 100 may take the form of a piezoelectric crystal mounted to the interior of the can of the pacemaker 10. Activity circuit 400 generates a signal or "count" each time the signal from the sensor 100 exceeds a certain optimal sensing threshold level. In prior art pacemakers of this general type, the threshold for the sensor 100 has typically been externally programmable to vary the amount of physical exertion required in order to trigger an output from the activity circuit 400. Alternatively, other sensors and sensor processing circuitry could be used in conjunction with the present invention so long as the sensor processing circuitry produces an output indicative of the patient' s metabolic demand for oxygenated blood. The output from activity circuit 400 is supplied to the logic and timing circuitry of digital controller/timer circuit 190 for altering the escape interval. The details of the implementation of the escape interval alteration circuitry are not provided since they are believed to be within the skill of pacemaker designers. FIG. 3 is a functional block diagram of the activity circuit 400 illustrated in FIG. 2. This diagram illustrates the functional interface between the major circuit blocks in the activity circuit 400 and the inputs and outputs to the remainder of the pacemaker 10 circuitry. As stated above, other activity processing circuitry could be used with the present invention so long as the activity processing circuitry produces an output indicative of the patient's metabolic demand for oxygenated blood. In the embodiment disclosed in FIGS. 2 and 3, the sensor output from activity sensor 100 is applied to sensor input 402 of amplifier 414 depicted in FIG. 3. Amplifier 414 has a programmable gain via multiple conductor signal path 404, to compensate for variations in activity sensor 100 output which may occur until the build process has stabilized and is completely characterized. The output from amplifier 414 is cascaded via ampout line 430 to a low-pass filter 416. Filter 416 is an inverting, adjustable gain, offset-compensated, low-pass filter stage which drives a peak detector 418. The gain of filter 416 is a function of programmable activity threshold settings whereby activity threshold settings are adjusted via multiple conductor signal path 406 which interfaces directly with digital/controller timer circuit 190. Detector block 418 is a peak detector circuit with 100% hysteresis to discriminate against large spurious type inputs and to enhance detector 418 sensitivity to periodic input signals. Clock circuit 420 provides the timing necessary for amplifier 414, filter 416 and detector 418 to interface in synchrony with other sections of input/output circuit 140 and microcomputer circuit 150. Power for amplifier 414, filter 416, detector 418 and power-on-reset (VDDPOR) 424 blocks is provided by bias circuit block 422. Turning now to FIG. 4, a flow diagram is shown which illustrates an automatic activity threshold adjusting algorithm in accordance with the presently disclosed embodiment of the invention. The algorithm of FIG. 4 begins by manually programming predetermined minimum and maximum zero activity time differences where zero activity time difference is the last zero activity time measurement minus the zero activity long term time average, and upon automatically querying patient activity counts as represented by block 500. Upon querying patient activity, digital controller/timer circuit 190 automatically prompts microcomputer circuit 150 to calculate and update a long term average time in which there has been an absence of patient activity, as represented in block 502. It is also understood that an absolute value representative of long term average zero activity time as represented in block 502 could also be a ratio such as a percentage represented by comparing short term zero activity time with long term average zero activity time. Immediately following the update of long term average time of zero activity, microcomputer circuit 150 interrogates the most recent query of patient activity to determine if there has been an absence of patient activity. This interrogation is represented in decision block 504. Following each query of patient activity in which there is an absence of activity as indicated by zero activity counts, the microcomputer increments a value stored in RAM circuit 166 or RAM/ROM unit 172 which represents a total cumulative time in which there is an absence of patient activity as shown in block 506. If the most recent query indicates that patient activity has occurred due to the presence of activity counts, the total cumulative time representing zero patient activity is not incremented, and the algorithm repeats itself by continuing to query activity sensor 100 as represented in block 500. This process will continue until microcomputer 150 determines that an activity threshold timer time-out has occurred as represented in block 508. As indicated above, in block 508 of FIG. 4, microcomputer 150 determines whether an activity threshold timer time-out has occurred. In the presently disclosed embodiment of the invention, it is contemplated that an activity threshold timer time-out will occur approximately every 24-hours, although time-out periods of any duration, ranging from one cardiac cycle to a month, may be employed. Subsequent to an activity threshold timer time-out, microcomputer circuit 150 calculates a time difference as represented in block 510. This time difference is equal to the last updated total cumulative zero activity time previously calculated in block 506 minus the long term zero activity time average previously calculated in block 502. Next, in decision block 512, a determination by microcomputer circuit 150 is made whether the time difference computed by microcomputer circuit 150 in block 510 is less than the predetermined minimum zero activity time difference value manually programmed in block 500. If so, microcomputer circuit 150 automatically increases the activity threshold as shown in block 516, toward its optimal value by prompting digital/controller timer circuit 190 to adjust the threshold level of activity circuit 400 accordingly. Activity threshold adjustments for activity circuit 400 are accomplished by enabling different combinations of threshold setting inputs to filter 416 via multiple conductor signal path 406. This enabling function is performed by digital/controller timer circuit 190 setting the state of each conductor within multiple conductor signal path 406, high or low, at the direction of microcomputer circuit 150. When the activity threshold is increased, activity circuit 400 becomes less sensitive to signals received from activity sensor 100, and less activity sensor 100 signals are likely to exceed the threshold level. If, however, the time difference value in block 510 exceeds the predetermined and manually programmed maximum zero activity time difference value in block 500 as represented in block 514, then the activity threshold level of activity circuit 400 is decreased toward its optimal value as represented in block 520. When the activity threshold is decreased, activity circuit 400 becomes more sensitive to signals received from activity sensor 100 and more activity sensor 100 signals are likely to exceed the threshold level. If the time difference value in block 510 is not less than the predetermined minimum zero activity time difference value in block 500 or greater than the predetermined maximum zero activity time difference value in block 500 as represented by decision blocks 512 and 514 respectively, then the activity threshold level of activity circuit 400 is already at its optimal value and no adjustment of activity threshold level transpires as represented in block 518. Following any adjustment of the activity threshold level, or in the alternative, a decision to maintain the activity threshold level at its present value, microcomputer circuit 150 in conjunction with input/output circuit 140 resets the zero activity time value stored in RAM 166 or RAM/ROM 172 and the activity threshold timeout timer as represented in block 522. This process for automatically and continually adjusting the activity threshold level toward its optimal value is repeated in perpetuity without any further programming time commitment by a physician. Thus, activity threshold will remain optimal, independent of the location of the pacemaker which may change as a result of subtle movements in the pocket, the IPG pocket characteristics, the patient's tissue composition, or the types of patient activities and vibrational/environmental factors. The algorithm depicted in FIG. 5 is an alternative embodiment, whereby steps 602 through 606 are carried out in the same manner as in the algorithm of FIG. 4. In this embodiment however, the algorithm begins by manually programming predetermined minimum and maximum time values, MINTIME and MAXTIME respectively, and also upon automatically querying patient activity counts as represented by block 600. From block 606, if an activity threshold timer time-out has not occurred, flow returns to block 600, as in the algorithm of FIG. 4. If an activity threshold time time-out has occurred, the comparison in block 608 is made, and the activity threshold is either increased, left the same, or decreased, as represented in blocks 612, 614 and 616 respectively. Thereafter, the activity threshold timer and zero activity time value are reset, as represented in block 618. The process for automatically and continually adjusting the activity threshold level toward its optimal value presented in this embodiment is analogous to the embodiment depicted in FIG. 4, whereby the optimization process repeats itself in perpetuity without any further programming time commitment by a physician. FIG. 6 depicts another alternative embodiment, whereby the process for automatically and continually adjusting the activity threshold level toward its optimal value is analogous to the embodiment depicted in FIG. 4 with one exception. This embodiment periodically updates the absolute value of long term zero activity time average as illustrated by block 708, whenever an independent long term average timer times out in block 706, whereas the embodiment illustrated in FIG. 4 updates the absolute value of long term zero activity time average continuously. From the foregoing detailed descriptions of particular embodiments of the invention, it should be apparent that a pacemaker has been disclosed which is provided with the capability of automatically adjusting the activity threshold of its activity sensing circuitry in order to minimize the probability of oversensing and undersensing on an on-going basis without requiring the attention of a physician. While particular embodiments of the present invention have been described herein in detail, it is to be understood that various alterations, modifications, and substitutions can be made therein without departing from the spirit and scope of the present invention, as defined in the claims, which follow. For example, it is contemplated by the inventors that timers and counters, such as those used for logging patient zero activity time and patient activity counts respectively, could be implemented by either software, hardware, or a combination of both. | Summary: A pacemaker capable of automatically adjusting the activity threshold setting of its activity sensor signal processing circuitry to its optimal value is disclosed. In one embodiment, the pacemaker maintains a running average of zero activity time and a cumulative summation of zero activity time over a predetermined history period. Periodically, the pacemaker computes a time difference between the running average of zero activity time and the cumulative summation of zero activity time, and adjusts the activity threshold of the activity sensor signal processing circuitry according to this computation. By basing the adjustment of activity threshold on a long-term average of zero activity time, the effects of cycle-to-cycle variation in sensed zero activity time are minimized. In another embodiment, the pacemaker periodically computes a time difference between a preprogrammed margin value and the cumulative summation of zero activity time. If the time difference is less than the predetermined margin, the activity threshold is increased. If the time difference is greater than the predetermined margin, the activity threshold is decreased. | 5,864 | 222 | big_patent | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Retirement Savings and Security Act of 2002''. SEC. 2. ACCELERATION OF INCREASES IN IRA CONTRIBUTION LIMIT. (a) Deductible Amount.--Subparagraph (A) of section 219(b)(5) of the Internal Revenue Code of 1986 is amended to read as follows: ``(A) In general.--The deductible amount shall be $5,000.''. (b) Catch-Up Amount.--Subparagraph (B) of section 219(b)(5) of such Code is amended to read as follows: ``(B) Catch-up contributions for individuals 50 or older.--In the case of an individual who has attained the age of 50 before the close of the taxable year, the dollar amount in effect under paragraph (1)(A) for such taxable year (determined without regard to this paragraph) shall be increased by $1,000.''. (c) Effective Date.--The amendments made by this section shall apply to taxable years beginning after December 31, 2002. SEC. 3. ACCELERATION OF SCHEDULED INCREASES IN PENSION PLAN CONTRIBUTION LIMITS. (a) Elective Deferrals.--Subparagraph (B) of section 402(g)(1) of the Internal Revenue Code of 1986 is amended by striking ``the amount determined'' and all that follows and inserting ``$15,000.''. (b) Deferred Compensation Plans of State and Local Governments and Tax-Exempt Organizations.--Subparagraph (A) of section 457(e)(15) of such Code is amended by striking ``the amount determined'' and all that follows and inserting ``$15,000.''. (c) Simple Retirement Accounts.--Clause (i) of section 408(p)(2)(E) of such Code is amended by striking ``the amount determined'' and all that follows and inserting ``$10,000.''. (d) Catch-Up Contributions.--Subparagraph (B) of section 414(v)(2) of such Code is amended-- (1) in clause (i) by striking ``determined'' and all that follows and inserting ``$5,000.'', and (2) in clause (ii) by striking ``determined'' and all that follows and inserting ``$2,500.''. (e) Effective Date.--The amendments made by this section shall apply to years beginning after December 31, 2002. SEC. 4. SIMPLIFICATION AND UPDATING OF THE MINIMUM DISTRIBUTION RULES. (a) Required Distributions.-- (1) Increase in age for required beginning date.-- Subparagraphs (C)(i)(I) and (C)(ii)(I) of section 401(a)(9) of the Internal Revenue Code of 1986 are each amended by striking ``age 70\1/2\'' and inserting ``the applicable age''. (2) Applicable age.--Subparagraph (C) of section 401(a)(9) of such Code is amended by inserting at the end the following new clause: ``(v) Applicable age.--The applicable age shall be determined in accordance with the following table: ``Calendar year: Applicable age is: 2003 and 2004.......................... 73 2005 and 2006.......................... 74 2007 and thereafter.................... 75.'' (3) Spouse beneficiaries.--Subclause (I) of section 401(a)(9)(B)(iv) is amended by striking ``age 70\1/2\'' and inserting ``the applicable age''. (4) Actuarial adjustment of benefit under defined benefit plan.--Clause (iii) of section 401(a)(9)(C) of such Code is amended to read as follows: ``(iii) Actuarial adjustment.-- ``(I) In general.--In the case of a defined benefit plan, an employee's accrued benefit shall be actuarially increased to take into account the period after the applicable date during which the employee was not eligible to receive any benefits under the plan. ``(II) Applicable date.--For purposes of clause (I), the term `applicable date' means the April 1st following the calendar year in which the employee attains age 70\1/2\.''. (b) Effective Date.-- (1) In general.--The amendments made by this section shall apply to years beginning after December 31, 2002. (2) Transition.--A plan shall not be treated as failing to meet the requirements of section 401(a)(9) of the Internal Revenue Code of 1986 merely because, in years beginning after December 31, 2002, no distribution is made to an employee before the employee's required beginning date, as determined in accordance with the amendments made by this section. | Title: To amend the Internal Revenue Code of 1986 to accelerate the increases in contribution limits to retirement plans and to increase the required beginning date for distributions from qualified plans Summary: Retirement Savings and Security Act of 2002 - Amends the Internal Revenue Code to accelerate, to 2003, increases to: (1) the $5,000 deductible IRA limit; and (2) the $15,000 elective deferral and the State, local, and tax-exempt organizations' plan limits, the $10,000 SIMPLE plan limit, and catch-up contribution limits.Increases incrementally, from the current of age 70 1/2 to age 75 starting in 2007, the required beginning date for distributions from qualified plans. | 1,268 | 185 | billsum | en |
Summarize: Gilbert Osmond came to see Isabel again; that is he came to Palazzo Crescentini. He had other friends there as well, and to Mrs. Touchett and Madame Merle he was always impartially civil; but the former of these ladies noted the fact that in the course of a fortnight he called five times, and compared it with another fact that she found no difficulty in remembering. Two visits a year had hitherto constituted his regular tribute to Mrs. Touchett's worth, and she had never observed him select for such visits those moments, of almost periodical recurrence, when Madame Merle was under her roof. It was not for Madame Merle that he came; these two were old friends and he never put himself out for her. He was not fond of Ralph--Ralph had told her so--and it was not supposable that Mr. Osmond had suddenly taken a fancy to her son. Ralph was imperturbable--Ralph had a kind of loose-fitting urbanity that wrapped him about like an ill-made overcoat, but of which he never divested himself; he thought Mr. Osmond very good company and was willing at any time to look at him in the light of hospitality. But he didn't flatter himself that the desire to repair a past injustice was the motive of their visitor's calls; he read the situation more clearly. Isabel was the attraction, and in all conscience a sufficient one. Osmond was a critic, a student of the exquisite, and it was natural he should be curious of so rare an apparition. So when his mother observed to him that it was plain what Mr. Osmond was thinking of, Ralph replied that he was quite of her opinion. Mrs. Touchett had from far back found a place on her scant list for this gentleman, though wondering dimly by what art and what process--so negative and so wise as they were--he had everywhere effectively imposed himself. As he had never been an importunate visitor he had had no chance to be offensive, and he was recommended to her by his appearance of being as well able to do without her as she was to do without him--a quality that always, oddly enough, affected her as providing ground for a relation with her. It gave her no satisfaction, however, to think that he had taken it into his head to marry her niece. Such an alliance, on Isabel's part, would have an air of almost morbid perversity. Mrs. Touchett easily remembered that the girl had refused an English peer; and that a young lady with whom Lord Warburton had not successfully wrestled should content herself with an obscure American dilettante, a middle-aged widower with an uncanny child and an ambiguous income, this answered to nothing in Mrs. Touchett's conception of success. She took, it will be observed, not the sentimental, but the political, view of matrimony--a view which has always had much to recommend it. "I trust she won't have the folly to listen to him," she said to her son; to which Ralph replied that Isabel's listening was one thing and Isabel's answering quite another. He knew she had listened to several parties, as his father would have said, but had made them listen in return; and he found much entertainment in the idea that in these few months of his knowing her he should observe a fresh suitor at her gate. She had wanted to see life, and fortune was serving her to her taste; a succession of fine gentlemen going down on their knees to her would do as well as anything else. Ralph looked forward to a fourth, a fifth, a tenth besieger; he had no conviction she would stop at a third. She would keep the gate ajar and open a parley; she would certainly not allow number three to come in. He expressed this view, somewhat after this fashion, to his mother, who looked at him as if he had been dancing a jig. He had such a fanciful, pictorial way of saying things that he might as well address her in the deaf-mute's alphabet. "I don't think I know what you mean," she said; "you use too many figures of speech; I could never understand allegories. The two words in the language I most respect are Yes and No. If Isabel wants to marry Mr. Osmond she'll do so in spite of all your comparisons. Let her alone to find a fine one herself for anything she undertakes. I know very little about the young man in America; I don't think she spends much of her time in thinking of him, and I suspect he has got tired of waiting for her. There's nothing in life to prevent her marrying Mr. Osmond if she only looks at him in a certain way. That's all very well; no one approves more than I of one's pleasing one's self. But she takes her pleasure in such odd things; she's capable of marrying Mr. Osmond for the beauty of his opinions or for his autograph of Michael Angelo. She wants to be disinterested: as if she were the only person who's in danger of not being so! Will HE be so disinterested when he has the spending of her money? That was her idea before your father's death, and it has acquired new charms for her since. She ought to marry some one of whose disinterestedness she shall herself be sure; and there would be no such proof of that as his having a fortune of his own." "My dear mother, I'm not afraid," Ralph answered. "She's making fools of us all. She'll please herself, of course; but she'll do so by studying human nature at close quarters and yet retaining her liberty. She has started on an exploring expedition, and I don't think she'll change her course, at the outset, at a signal from Gilbert Osmond. She may have slackened speed for an hour, but before we know it she'll be steaming away again. Excuse another metaphor." Mrs. Touchett excused it perhaps, but was not so much reassured as to withhold from Madame Merle the expression of her fears. "You who know everything," she said, "you must know this: whether that curious creature's really making love to my niece." "Gilbert Osmond?" Madame Merle widened her clear eyes and, with a full intelligence, "Heaven help us," she exclaimed, "that's an idea!" "Hadn't it occurred to you?" "You make me feel an idiot, but I confess it hadn't. I wonder," she added, "if it has occurred to Isabel." "Oh, I shall now ask her," said Mrs. Touchett. Madame Merle reflected. "Don't put it into her head. The thing would be to ask Mr. Osmond." "I can't do that," said Mrs. Touchett. "I won't have him enquire of me--as he perfectly may with that air of his, given Isabel's situation--what business it is of mine." "I'll ask him myself," Madame Merle bravely declared. "But what business--for HIM--is it of yours?" "It's being none whatever is just why I can afford to speak. It's so much less my business than any one's else that he can put me off with anything he chooses. But it will be by the way he does this that I shall know." "Pray let me hear then," said Mrs. Touchett, "of the fruits of your penetration. If I can't speak to him, however, at least I can speak to Isabel." Her companion sounded at this the note of warning. "Don't be too quick with her. Don't inflame her imagination." "I never did anything in life to any one's imagination. But I'm always sure of her doing something--well, not of MY kind." "No, you wouldn't like this," Madame Merle observed without the point of interrogation. "Why in the world should I, pray? Mr. Osmond has nothing the least solid to offer." Again Madame Merle was silent while her thoughtful smile drew up her mouth even more charmingly than usual toward the left corner. "Let us distinguish. Gilbert Osmond's certainly not the first comer. He's a man who in favourable conditions might very well make a great impression. He has made a great impression, to my knowledge, more than once." "Don't tell me about his probably quite cold-blooded love-affairs; they're nothing to me!" Mrs. Touchett cried. "What you say's precisely why I wish he would cease his visits. He has nothing in the world that I know of but a dozen or two of early masters and a more or less pert little daughter." "The early masters are now worth a good deal of money," said Madame Merle, "and the daughter's a very young and very innocent and very harmless person." "In other words she's an insipid little chit. Is that what you mean? Having no fortune she can't hope to marry as they marry here; so that Isabel will have to furnish her either with a maintenance or with a dowry." "Isabel probably wouldn't object to being kind to her. I think she likes the poor child." "Another reason then for Mr. Osmond's stopping at home! Otherwise, a week hence, we shall have my niece arriving at the conviction that her mission in life's to prove that a stepmother may sacrifice herself--and that, to prove it, she must first become one." "She would make a charming stepmother," smiled Madame Merle; "but I quite agree with you that she had better not decide upon her mission too hastily. Changing the form of one's mission's almost as difficult as changing the shape of one's nose: there they are, each, in the middle of one's face and one's character--one has to begin too far back. But I'll investigate and report to you." All this went on quite over Isabel's head; she had no suspicions that her relations with Mr. Osmond were being discussed. Madame Merle had said nothing to put her on her guard; she alluded no more pointedly to him than to the other gentlemen of Florence, native and foreign, who now arrived in considerable numbers to pay their respects to Miss Archer's aunt. Isabel thought him interesting--she came back to that; she liked so to think of him. She had carried away an image from her visit to his hill-top which her subsequent knowledge of him did nothing to efface and which put on for her a particular harmony with other supposed and divined things, histories within histories: the image of a quiet, clever, sensitive, distinguished man, strolling on a moss-grown terrace above the sweet Val d'Arno and holding by the hand a little girl whose bell-like clearness gave a new grace to childhood. The picture had no flourishes, but she liked its lowness of tone and the atmosphere of summer twilight that pervaded it. It spoke of the kind of personal issue that touched her most nearly; of the choice between objects, subjects, contacts--what might she call them?--of a thin and those of a rich association; of a lonely, studious life in a lovely land; of an old sorrow that sometimes ached to-day; of a feeling of pride that was perhaps exaggerated, but that had an element of nobleness; of a care for beauty and perfection so natural and so cultivated together that the career appeared to stretch beneath it in the disposed vistas and with the ranges of steps and terraces and fountains of a formal Italian garden--allowing only for arid places freshened by the natural dews of a quaint half-anxious, half-helpless fatherhood. At Palazzo Crescentini Mr. Osmond's manner remained the same; diffident at first--oh self-conscious beyond doubt! and full of the effort (visible only to a sympathetic eye) to overcome this disadvantage; an effort which usually resulted in a great deal of easy, lively, very positive, rather aggressive, always suggestive talk. Mr. Osmond's talk was not injured by the indication of an eagerness to shine; Isabel found no difficulty in believing that a person was sincere who had so many of the signs of strong conviction--as for instance an explicit and graceful appreciation of anything that might be said on his own side of the question, said perhaps by Miss Archer in especial. What continued to please this young woman was that while he talked so for amusement he didn't talk, as she had heard people, for "effect." He uttered his ideas as if, odd as they often appeared, he were used to them and had lived with them; old polished knobs and heads and handles, of precious substance, that could be fitted if necessary to new walking-sticks--not switches plucked in destitution from the common tree and then too elegantly waved about. One day he brought his small daughter with him, and she rejoiced to renew acquaintance with the child, who, as she presented her forehead to be kissed by every member of the circle, reminded her vividly of an ingenue in a French play. Isabel had never seen a little person of this pattern; American girls were very different--different too were the maidens of England. Pansy was so formed and finished for her tiny place in the world, and yet in imagination, as one could see, so innocent and infantine. She sat on the sofa by Isabel; she wore a small grenadine mantle and a pair of the useful gloves that Madame Merle had given her--little grey gloves with a single button. She was like a sheet of blank paper--the ideal jeune fille of foreign fiction. Isabel hoped that so fair and smooth a page would be covered with an edifying text. The Countess Gemini also came to call upon her, but the Countess was quite another affair. She was by no means a blank sheet; she had been written over in a variety of hands, and Mrs. Touchett, who felt by no means honoured by her visit, pronounced that a number of unmistakeable blots were to be seen upon her surface. The Countess gave rise indeed to some discussion between the mistress of the house and the visitor from Rome, in which Madame Merle (who was not such a fool as to irritate people by always agreeing with them) availed herself felicitously enough of that large licence of dissent which her hostess permitted as freely as she practised it. Mrs. Touchett had declared it a piece of audacity that this highly compromised character should have presented herself at such a time of day at the door of a house in which she was esteemed so little as she must long have known herself to be at Palazzo Crescentini. Isabel had been made acquainted with the estimate prevailing under that roof: it represented Mr. Osmond's sister as a lady who had so mismanaged her improprieties that they had ceased to hang together at all--which was at the least what one asked of such matters--and had become the mere floating fragments of a wrecked renown, incommoding social circulation. She had been married by her mother--a more administrative person, with an appreciation of foreign titles which the daughter, to do her justice, had probably by this time thrown off--to an Italian nobleman who had perhaps given her some excuse for attempting to quench the consciousness of outrage. The Countess, however, had consoled herself outrageously, and the list of her excuses had now lost itself in the labyrinth of her adventures. Mrs. Touchett had never consented to receive her, though the Countess had made overtures of old. Florence was not an austere city; but, as Mrs. Touchett said, she had to draw the line somewhere. Madame Merle defended the luckless lady with a great deal of zeal and wit. She couldn't see why Mrs. Touchett should make a scapegoat of a woman who had really done no harm, who had only done good in the wrong way. One must certainly draw the line, but while one was about it one should draw it straight: it was a very crooked chalk-mark that would exclude the Countess Gemini. In that case Mrs. Touchett had better shut up her house; this perhaps would be the best course so long as she remained in Florence. One must be fair and not make arbitrary differences: the Countess had doubtless been imprudent, she had not been so clever as other women. She was a good creature, not clever at all; but since when had that been a ground of exclusion from the best society? For ever so long now one had heard nothing about her, and there could be no better proof of her having renounced the error of her ways than her desire to become a member of Mrs. Touchett's circle. Isabel could contribute nothing to this interesting dispute, not even a patient attention; she contented herself with having given a friendly welcome to the unfortunate lady, who, whatever her defects, had at least the merit of being Mr. Osmond's sister. As she liked the brother Isabel thought it proper to try and like the sister: in spite of the growing complexity of things she was still capable of these primitive sequences. She had not received the happiest impression of the Countess on meeting her at the villa, but was thankful for an opportunity to repair the accident. Had not Mr. Osmond remarked that she was a respectable person? To have proceeded from Gilbert Osmond this was a crude proposition, but Madame Merle bestowed upon it a certain improving polish. She told Isabel more about the poor Countess than Mr. Osmond had done, and related the history of her marriage and its consequences. The Count was a member of an ancient Tuscan family, but of such small estate that he had been glad to accept Amy Osmond, in spite of the questionable beauty which had yet not hampered her career, with the modest dowry her mother was able to offer--a sum about equivalent to that which had already formed her brother's share of their patrimony. Count Gemini since then, however, had inherited money, and now they were well enough off, as Italians went, though Amy was horribly extravagant. The Count was a low-lived brute; he had given his wife every pretext. She had no children; she had lost three within a year of their birth. Her mother, who had bristled with pretensions to elegant learning and published descriptive poems and corresponded on Italian subjects with the English weekly journals, her mother had died three years after the Countess's marriage, the father, lost in the grey American dawn of the situation, but reputed originally rich and wild, having died much earlier. One could see this in Gilbert Osmond, Madame Merle held--see that he had been brought up by a woman; though, to do him justice, one would suppose it had been by a more sensible woman than the American Corinne, as Mrs. Osmond had liked to be called. She had brought her children to Italy after her husband's death, and Mrs. Touchett remembered her during the year that followed her arrival. She thought her a horrible snob; but this was an irregularity of judgement on Mrs. Touchett's part, for she, like Mrs. Osmond, approved of political marriages. The Countess was very good company and not really the featherhead she seemed; all one had to do with her was to observe the simple condition of not believing a word she said. Madame Merle had always made the best of her for her brother's sake; he appreciated any kindness shown to Amy, because (if it had to be confessed for him) he rather felt she let down their common name. Naturally he couldn't like her style, her shrillness, her egotism, her violations of taste and above all of truth: she acted badly on his nerves, she was not HIS sort of woman. What was his sort of woman? Oh, the very opposite of the Countess, a woman to whom the truth should be habitually sacred. Isabel was unable to estimate the number of times her visitor had, in half an hour, profaned it: the Countess indeed had given her an impression of rather silly sincerity. She had talked almost exclusively about herself; how much she should like to know Miss Archer; how thankful she should be for a real friend; how base the people in Florence were; how tired she was of the place; how much she should like to live somewhere else--in Paris, in London, in Washington; how impossible it was to get anything nice to wear in Italy except a little old lace; how dear the world was growing everywhere; what a life of suffering and privation she had led. Madame Merle listened with interest to Isabel's account of this passage, but she had not needed it to feel exempt from anxiety. On the whole she was not afraid of the Countess, and she could afford to do what was altogether best--not to appear so. Isabel had meanwhile another visitor, whom it was not, even behind her back, so easy a matter to patronise. Henrietta Stackpole, who had left Paris after Mrs. Touchett's departure for San Remo and had worked her way down, as she said, through the cities of North Italy, reached the banks of the Arno about the middle of May. Madame Merle surveyed her with a single glance, took her in from head to foot, and after a pang of despair determined to endure her. She determined indeed to delight in her. She mightn't be inhaled as a rose, but she might be grasped as a nettle. Madame Merle genially squeezed her into insignificance, and Isabel felt that in foreseeing this liberality she had done justice to her friend's intelligence. Henrietta's arrival had been announced by Mr. Bantling, who, coming down from Nice while she was at Venice, and expecting to find her in Florence, which she had not yet reached, called at Palazzo Crescentini to express his disappointment. Henrietta's own advent occurred two days later and produced in Mr. Bantling an emotion amply accounted for by the fact that he had not seen her since the termination of the episode at Versailles. The humorous view of his situation was generally taken, but it was uttered only by Ralph Touchett, who, in the privacy of his own apartment, when Bantling smoked a cigar there, indulged in goodness knew what strong comedy on the subject of the all-judging one and her British backer. This gentleman took the joke in perfectly good part and candidly confessed that he regarded the affair as a positive intellectual adventure. He liked Miss Stackpole extremely; he thought she had a wonderful head on her shoulders, and found great comfort in the society of a woman who was not perpetually thinking about what would be said and how what she did, how what they did--and they had done things!--would look. Miss Stackpole never cared how anything looked, and, if she didn't care, pray why should he? But his curiosity had been roused; he wanted awfully to see if she ever WOULD care. He was prepared to go as far as she--he didn't see why he should break down first. Henrietta showed no signs of breaking down. Her prospects had brightened on her leaving England, and she was now in the full enjoyment of her copious resources. She had indeed been obliged to sacrifice her hopes with regard to the inner life; the social question, on the Continent, bristled with difficulties even more numerous than those she had encountered in England. But on the Continent there was the outer life, which was palpable and visible at every turn, and more easily convertible to literary uses than the customs of those opaque islanders. Out of doors in foreign lands, as she ingeniously remarked, one seemed to see the right side of the tapestry; out of doors in England one seemed to see the wrong side, which gave one no notion of the figure. The admission costs her historian a pang, but Henrietta, despairing of more occult things, was now paying much attention to the outer life. She had been studying it for two months at Venice, from which city she sent to the Interviewer a conscientious account of the gondolas, the Piazza, the Bridge of Sighs, the pigeons and the young boatman who chanted Tasso. The Interviewer was perhaps disappointed, but Henrietta was at least seeing Europe. Her present purpose was to get down to Rome before the malaria should come on--she apparently supposed that it began on a fixed day; and with this design she was to spend at present but few days in Florence. Mr. Bantling was to go with her to Rome, and she pointed out to Isabel that as he had been there before, as he was a military man and as he had had a classical education--he had been bred at Eton, where they study nothing but Latin and Whyte-Melville, said Miss Stackpole--he would be a most useful companion in the city of the Caesars. At this juncture Ralph had the happy idea of proposing to Isabel that she also, under his own escort, should make a pilgrimage to Rome. She expected to pass a portion of the next winter there--that was very well; but meantime there was no harm in surveying the field. There were ten days left of the beautiful month of May--the most precious month of all to the true Rome-lover. Isabel would become a Rome-lover; that was a foregone conclusion. She was provided with a trusty companion of her own sex, whose society, thanks to the fact of other calls on this lady's attention, would probably not be oppressive. Madame Merle would remain with Mrs. Touchett; she had left Rome for the summer and wouldn't care to return. She professed herself delighted to be left at peace in Florence; she had locked up her apartment and sent her cook home to Palestrina. She urged Isabel, however, to assent to Ralph's proposal, and assured her that a good introduction to Rome was not a thing to be despised. Isabel in truth needed no urging, and the party of four arranged its little journey. Mrs. Touchett, on this occasion, had resigned herself to the absence of a duenna; we have seen that she now inclined to the belief that her niece should stand alone. One of Isabel's preparations consisted of her seeing Gilbert Osmond before she started and mentioning her intention to him. "I should like to be in Rome with you," he commented. "I should like to see you on that wonderful ground." She scarcely faltered. "You might come then." "But you'll have a lot of people with you." "Ah," Isabel admitted, "of course I shall not be alone." For a moment he said nothing more. "You'll like it," he went on at last. "They've spoiled it, but you'll rave about it." "Ought I to dislike it because, poor old dear--the Niobe of Nations, you know--it has been spoiled?" she asked. "No, I think not. It has been spoiled so often," he smiled. "If I were to go, what should I do with my little girl?" "Can't you leave her at the villa?" "I don't know that I like that--though there's a very good old woman who looks after her. I can't afford a governess." "Bring her with you then," said Isabel promptly. Mr. Osmond looked grave. "She has been in Rome all winter, at her convent; and she's too young to make journeys of pleasure." "You don't like bringing her forward?" Isabel enquired. "No, I think young girls should be kept out of the world." "I was brought up on a different system." "You? Oh, with you it succeeded, because you--you were exceptional." "I don't see why," said Isabel, who, however, was not sure there was not some truth in the speech. Mr. Osmond didn't explain; he simply went on: "If I thought it would make her resemble you to join a social group in Rome I'd take her there to-morrow." "Don't make her resemble me," said Isabel. "Keep her like herself." "I might send her to my sister," Mr. Osmond observed. He had almost the air of asking advice; he seemed to like to talk over his domestic matters with Miss Archer. "Yes," she concurred; "I think that wouldn't do much towards making her resemble me!" After she had left Florence Gilbert Osmond met Madame Merle at the Countess Gemini's. There were other people present; the Countess's drawing-room was usually well filled, and the talk had been general, but after a while Osmond left his place and came and sat on an ottoman half-behind, half-beside Madame Merle's chair. "She wants me to go to Rome with her," he remarked in a low voice. "To go with her?" "To be there while she's there. She proposed it. "I suppose you mean that you proposed it and she assented." "Of course I gave her a chance. But she's encouraging--she's very encouraging." "I rejoice to hear it--but don't cry victory too soon. Of course you'll go to Rome." "Ah," said Osmond, "it makes one work, this idea of yours!" "Don't pretend you don't enjoy it--you're very ungrateful. You've not been so well occupied these many years." "The way you take it's beautiful," said Osmond. "I ought to be grateful for that." "Not too much so, however," Madame Merle answered. She talked with her usual smile, leaning back in her chair and looking round the room. "You've made a very good impression, and I've seen for myself that you've received one. You've not come to Mrs. Touchett's seven times to oblige me." "The girl's not disagreeable," Osmond quietly conceded. Madame Merle dropped her eye on him a moment, during which her lips closed with a certain firmness. "Is that all you can find to say about that fine creature?" "All? Isn't it enough? Of how many people have you heard me say more?" She made no answer to this, but still presented her talkative grace to the room. "You're unfathomable," she murmured at last. "I'm frightened at the abyss into which I shall have cast her." He took it almost gaily. "You can't draw back--you've gone too far." "Very good; but you must do the rest yourself." "I shall do it," said Gilbert Osmond. Madame Merle remained silent and he changed his place again; but when she rose to go he also took leave. Mrs. Touchett's victoria was awaiting her guest in the court, and after he had helped his friend into it he stood there detaining her. "You're very indiscreet," she said rather wearily; "you shouldn't have moved when I did." He had taken off his hat; he passed his hand over his forehead. "I always forget; I'm out of the habit." "You're quite unfathomable," she repeated, glancing up at the windows of the house, a modern structure in the new part of the town. He paid no heed to this remark, but spoke in his own sense. "She's really very charming. I've scarcely known any one more graceful." "It does me good to hear you say that. The better you like her the better for me." "I like her very much. She's all you described her, and into the bargain capable, I feel, of great devotion. She has only one fault." "What's that?" "Too many ideas." "I warned you she was clever." "Fortunately they're very bad ones," said Osmond. "Why is that fortunate?" "Dame, if they must be sacrificed!" Madame Merle leaned back, looking straight before her; then she spoke to the coachman. But her friend again detained her. "If I go to Rome what shall I do with Pansy?" "I'll go and see her," said Madame Merle. | Summary: Gilbert Osmond comes to the Palazzo Crescentini five times. Mrs. Touchett realizes he has never come more than twice in a single year and that since he cant possibly be interested in Madame Merle, he must be interested in Isabel. She asks Ralph about it and he says it is sure that Gilbert Osmond is interested in Isabel, but that they neednt worry since Isabel has higher plans than would be fulfilled by Osmond. Mrs. Touchett also confers with Madame Merle about it. Madame Merle acts as if the thought hasnt occurred to her, but says she will sound Gilbert Osmond out about it and advises Mrs. Touchett not to say anything to Isabel. Isabel, her own part, has developed her initial romantic image of Gilbert Osmond as a "quiet, clever, sensitive, distinguished man, who is living a"lonely, studious life in a lovely land, "picturesquely standing beside his remarkably innocent daughter. The Countess Gemini comes to call several times as well. Mrs. Touchett doesnt like to receive her since she is such a scandal to talk to. Madame Merle tries to soothe her about the Countess taking the latters part. By the way of doing this, she informs Isabel of Amy and Gilbert Osmonds parentage. Their mother had been a minor poet who moved to Italy with her two children after her husband,"originally rice and wild, "had died. Isabel tries to be kind to the Countess only because she likes Gilbert Osmond and wants to like his sister. Meanwhile, Henrietta Stackpole comes to Venice and spends time with Isabel. She has been having a wonderful time in France and is now proceeding through Italy. She is preceded by Mr. Bantling, who tells Ralph of his admiration for her and his determination to follow through with all that she allows him of her company just to see how far shell go. When Henrietta arrives, she proposes a trip to Rome. Ralph wants to go as well. The four of them leave together. Gilbert Osmond meets Madame Merle at the Countess Geminis house at one of her parties. He sits slightly behind and to the side of Madame Merle and they carry on a conversation in whispers, acting like they are not together. They discuss the idea of Madame Merles of getting him and Isabel Archer married. He tells her that the way she takes his attention to the younger woman is beautiful. He tells her Isabel is"not disagreeable. "She says he is"unfathomable "and that she is afraid"at the abyss into which shall have cast. "He gets up and leaves, but when she gets up to leave the house, he goes out with her. When they get outside and shes in her carriage, she scolds him with being so indiscreet. They continue their conversation. He tells her Isabel is very charming and graceful. Madame Merle says the more he likes Isabel, the better it is for her, Madame Merle. Gilbert says the only problem with Isabel is that she has too many ideas, but since theyre bad ideas, its not so bad, since they will have to be sacrificed. Their last words are about Pansy. Madame Merle says shell take care of Pansy while he goes to Rome. | 7,250 | 704 | booksum | en |
Summarize: BACKGROUND OF THE INVENTION The invention concerns a crop divider, provided at a lateral end of a transversely extending harvesting header or gatherer for dividing the crop to be gathered from the standing crop, and more particularly, a means of mounting such a divider. Crop dividers are well known and are used on a variety of harvesting machines. They are frequently used, for example, on cutterbar type harvesting platforms, the pointed ends of the dividers extending forwardly of the opposite ends of the cutterbar. Typically, dividers are pivotably mounted so that they are free to swing in a vertical arc in a fore-and-aft plane about a fixed single pivot point, to follow the contour of the ground. Some dividers are partially counter-balanced, for example, by springs. In typical operation, with the harvester gatherer or platform at operating height above the ground, the divider rides on the ground to guide or divide the crop into the gatherer, pivoting up and down to follow variations in the ground surface. Typically, the operating height of the harvester gatherer or platform is vertically adjustable, the operator setting the height according to crop or harvesting conditions. The angle of inclination of the divider in a fore-and-aft plane when operating in contact with the ground will, of course, vary and depend entirely on the operating height of the platform. When the crop divider is carried on a simple single pivot, the range of divider angle of inclination or attitude thus defined, may significantly exceed an optimum range for efficient operation of the divider or divider point in dividing and lifting crop. SUMMARY OF THE INVENTION Accordingly, it is an object of the invention to provide a mounting or support arrangement for a harvester crop divider so that it moves, in its working range, in a modified manner compared with the conventional simply pivoted crop divider. A more specific object of the invention is to provide a divider mounting arangement in which the divider effective pivot point and in which the change of angle of inclination of the divider point, for a given change in operating height of the platform, varies according to the operating height of the platform above the ground. According to the invention, the crop divider is loosely supported in a generally fore-and-aft extending guide carried by the frame of the harvester platform or gatherer. An elongated fore-and-aft extending frame member or spine of the crop divider extends rearwardly through the guide. The form of the guide prevents rotation of the crop divider about its fore-and-aft axis, but permits pivoting and translational movement in an upright fore-and-aft plane. The pivot point of the divider with respect to the gatherer or platform frame may be said to be undefined, but engagement of the divider frame member or spine with the guide, in operation, establishes at least a pair of effective pivot points as the divider moves through its operating range. Movement of the crop divider frame member or spine within the guide is unrestricted, except for the provision of a stop arrangement, effective between the spine and the guide, for maintaining an approximately constant fore-and-aft disposition of the crop divider with respect to the guide. In a preferred embodiment of the invention, the harvester gatherer or platform frame includes at each of its opposite ends a pair of vertical slots, spaced apart fore-and-aft, with the front slot rearward of the divider center of gravity. The slots are disposed and the spine is formed, so that the harvester platform elevated to a transport position, the crop divider point hangs down under the action of gravity with the spine "stopped" by the bottom of the front slot and the top of the rear slot. As the platform is lowered towards operating height, the crop divider point engages the ground and the spine effectively pivots about the bottom of the forward slot. As the platform is lowered further, the spine rocks back in the guide, eventually lifting from the bottom of the forward slot and engaging the bottom of the rearward slot, which thus becomes the effective pivot point. Upward movement of the divider point with respect to the platform frame is finally limited by the spine engaging the top of the front slot and the bottom of the rear slot. Crop divider operating characteristics deriving from a divider mounting arrangement according to the invention are generally suitable for all operating conditions and include some significant and useful differences from those of conventional dividers. For example, upon first contact of the divider point with the ground, as the platform is lowered towards operating height, the effective pivot point of the divider is relatively forward so that the unbalanced weight of the divider is relatively low. Thus, ground pressure is also relatively low so that the divider point readily pivots upwards as lowering of the platform continues, or as ground surface conditions require. Friction between the divider point and the ground is relatively low and the reduced ground pressure reduces the possibility of the point digging in in soft conditions. The relatively forward position of the divider effective pivot point, effective in the upper ranges of operating height, results in a desirable relatively rapid change in angle of inclination of the divider point for a given change in operating height, so that the divider point is rapidly rotated towards its optimum operating attitude as the platform is lowered. In the lower ranges of operating height, used, for example, in down grain crops or soybeans, the rearward pivot is in effect. The greater radius arm from divider point to pivot means that the point can ride up with relatively less change in attitude or angularity, so that the point can continue to get under and lift down crop--the forward working tip of the divider point is kept close to the ground. When an extra long divider is used, again as, for example, in harvesting soybeans or down or tangled crop, the possibility of translational movement of the spine within the guide becomes useful. If a ground-engaging surface or skid shoe on the bottom of the divider point is of significant length, the divider point may ride flat on the ground and rise up and down relative to the platform frame without substantial change in angularity or attitude. The "looseness" of the spine within the guide permits this mode of operation--equivalent to an effective pivot point at an infinite distance to the rear of the divider point. At low platform operating heights, with the divider point relatively flat on the ground, the effective rearward pivot point provided by the engagement of the spine with the rear slot, or the infinitely rearward effective pivot point of the floating mode, result in relatively greater pressure between the point and the ground, helping the point to hug the ground and get under the crop. In the flat attitude, there is less risk of the divider point digging into the ground. It is a further advantage of a divider arrangement according to the present invention, that for a given length of divider assembly, including particularly the length of the divider elongated frame or spine member, the simple structure of fore-and-aft apertures with opportunity for vertical movement in both, provides a greater total range of floating adjustment for the divider point than a conventional pivoted divider having a similar range-limiting front bracket or aperture but only a simple single fixed pivot rearward of that aperture. The potential for rearward disposition of the effective pivot point gives a divider mounting according to the invention some of the advantages and operating characteristics of a simply pivoted long divider but using a shorter, inherently more stable structure. In the invention, a simple structure provides not only a desirable floating range, but the mounting arrangement itself automatically provides stable support in transport, and there is no need for special stops or brackets. A further advantage of the invention is that, with adequate fore-and-aft spacing between the front and rear apertures or brackets, lateral stability of the divider is maintained without the need for additional stablizing structure. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a left front perspective view of a self-propelled combine harvester equipped with a cutting platform and having a pair of crop dividers according to the invention. FIG. 2 is an enlarged partial rear three-quarter perspective view of the outer side of the right-hand crop divider of the combine of FIG. 1. FIG. 3 is a semi-schematic elevation of a crop divider, according to the invention, with the divider point shown in a range of working positions. DESCRIPTION OF THE PREFERRED EMBODIMENT The invention is embodied in a self-propelled combine harvester 10, shown in FIG. 1. Apart from the invention, the harvester is generally conventional. It is propelled by powered front wheels 12 and steered by rear wheels 14. The forward mounted vertically adjustable header 16 carries a front mounted gatherer or harvesting device, in this case a cutting platform 18, feeding gathered material to a feeder house 20 for transmission to the body of the harvester for processing. The harvester is controlled from an operator's station 22. The cutting platform 18 has a material transfer floor or bottom 24, bordered by opposite fore-and-aft extending side walls, left- and right-hand, 26, 28, respectively, behind a conventional cutterbar 30. Crop dividers 31 and 32, left- and right-hand, respectively, extend forward from the side walls 26, 28, for dividing the crop and guiding a swath into engagement by the cutterbar 30. The dividers 31, 32, are generallly indentical but of opposite hand and only the right-hand divider 32, shown in FIG. 2, will be described in any detail. The divider 32 includes a fore-and-aft extending main frame member or spine 34, in this case of tubular construction, carrying a frusto-conical divider point 36, having a divider point nose or shoe 37. Attachment of the point 36 to the spine 34 is reinforced and made adjustable by adjustable brace 38, including suitable hardware 40. The dividing and deflecting function of the divider point 36 is extended by outer and inner elongated deflectors 42, 44, respectively. Deflector 42 is tied directly to the spine frame member 34, while the inner deflector 42 is braced by a strut 46. Spaced fore-and-aft, on the outside of the right-hand platform side wall 28, are a pair of guide brackets rear and front, 50, 52, respectively. The brackets each have generally vertically extending slots 54, 56, in the front and rear brackets respectively. The slots have opposite outer and inner upright edges, 58, 59 and 60, 61, respectively. Top and bottom portions of the slots are, front and rear respectively, top 62, 63 and bottom 64, 65. Preferably the slotted brackets 50, 52 are positioned so that the center of gravity of the divider 32 is forward of the front bracket 52. A stabilizing flange 70 abuts and is rigidly attached to the end of the spine tube 34. In assembly, the flange 70 is inserted in the rear slot 56, and insertion of locking element or retaining pin 72 secures the divider 32 against fore-and-aft movement, but permits the flange 70 to slide up and down in the rear slot 56, with the butt end of the spine tube 34 adjacent the forward face 73 of the rear bracket 50. The fit of the flange 70 in the slot 56 and its vertical extent prevent the divider 32 from rotating about its fore-and-aft axis. Preferably, the fit of the spine 34 in the front slot 54 and of the flange 70 in the rear slot 56 are such as to provide lateral stability for the divider 32. But, it desired, this function could be provided or assisted by additional guide surfaces (not shown) independent of the slots 54, 56. In operation, the handling of the combine harvester 10 is generally conventional. In transport the header 16 is usually fully elevated (not shown in the drawings), so that the divider 32 is clear of the ground and hangs in a "ready" position with the spine 34 resting on the bottom 64 of the front slot 54, and the flange 70 abutting the upper end 63 of the rear slot 56. The bottom 64 of the front slot 54 is inclined to form a vee with the upright outer edge 58. This cradles the spine 34 and reduces rattling of the divider during transport on rough terrain. As the header is lowered into operating position (similar to the position shown in FIG. 1), the shoe 37 of the divider point 36 contacts the ground and the point 36 moves upwards relative to the platform 18 to a first operating position indicated by numeral I in FIG. 3. Here the flange 70 has come away from its upper stop 63, and the spine 34 is pivoting on the lower edge 64 of the front slot. Especially if the front bracket 52 is relatively far ahead, the weight of the divider 32 will be distributed so that a significant portion of the weight of the divider is borne by the front bracket 52, thus reducing the ground pressure of the divider point 36, so that it may respond more readily to changes in field surface levels. As the cutting platform 18 is lowered still further, or as the divider rides up due to surface variations, the divider 32 may assume a position indicated at numeral II in FIG. 3, where the spine 34 is lifted from the bottom 64 of the front slot and is effectively pivoting, by means of flange 70, about the bottom end 65 of the rear slot. Ground pressure of the divider is thus relatively increased because of the rearward movement of the effective pivot point of the divider. Thus, during movement of the crop divider 32, from position I into position II, the effective pivot point travels or transfers from the front barcket 52 to the rear bracket 50. Momentarily, the pivot point for the divider is undefined and, at least in part of the range of operating height, because of the depth of the slots 54, 56, the divider is free to float in a translational mode as well as pivoting, with or without contact at the bottoms 64, 65 of the slots. Thus the advantages of a range of pivot or support arrangements is obtained with a simple, compact structure. The displacement of the crop divider 32 between the positions I and II of FIG. 3 corresponds to a typical operating range. An upward limiting position is illustrated at numeral III of FIG. 3. The spine 34 is prevented from further upward movement by having come into contact with the top 62 of the front slot and the bottom 65 of the rear slot. The free floating divider support arrangement of the invention has been illustrated in a self-propelled combine harvester application. But, of course, it may be used in other applications, such as the dividers of other harvesting machines, or even in such devices as a planter furrow marker, or in any application where it is useful for the ground pressure and pivoting characteristic of the ground-engaging portion of a device to be variable, at least in part, according to the operating position of the device relative to the frame which carries it. | Summary: A crop divider of the cutting platform of a self-propelled combine harvester is loosely supported for floating and rocking movement in a vertical fore-and-aft plane, by a rearwardly extending frame member or spine of the divider being inserted into and extending through a pair of vertically slotted brackets, spaced fore-and-aft. The pivot point for the divider is thus undefined and may move back and forth between the front and rear brackets according to the operating height of the cutting platform. | 3,636 | 121 | big_patent | en |
Summarize: Background: Federal Laws and Guidance Govern Use of Personal Information in Federal Agencies A core function of privacy officers is to ensure that their agencies are in compliance with federal laws. The major requirements for th protection of personal privacy by federal agencies come from two laws, the Privacy Act of 1974 and the E-Government Act of 2002. The Federal Information Security Management Act of 2002 (FISM A) also addresses the protection of personal information in the context of securing federal agency information and information systems. The Privacy Act places limitations on agencies’ collection, disclosure, and use of personal information maintained in s ystems of records. The act describes a “record” as any item, collection, or grouping of information about an individual that is maintained by a agency and contains his or her name or another personal identifier. It also defines “system of records” as a group of records under the control of any agency from which information is retrieved by the n name of the individual or by an individual identifier. The Privacy A requires that when agencies establish or make changes to a system of records, they must notify the public by a “system-of-records notice”: that is, a notice in the Federal Register identifying, amo other things, the type of data collected, the types of individuals about whom information is collected, the intended “routine” use data, and procedures that individuals can use to review and correct personal information. Among other provisions, the act also requires agencies to define and limit themselves to specific predefined purposes. For example, the act requires that to the greatest ext ent practicable, personal information should be collected directly from the subject individual when it may affect an individual’s rights or benefits under a federal program. The provisions of the Privacy Act are largely based on a set of principles for protecting the privacy and security of personal information, known as the Fair Information Practices, which w first proposed in 1973 by a U.S. government advisory committee; these principles were intended to address what the committee termed a poor level of protection afforded to privacy under contemporary law. Since that time, the Fair Information Pra ctices have been widely adopted as a standard benchmark for evaluating the adequacy of privacy protections. Attachment 2 contains a summary of the widely used version of the Fair Information Practices adopted by the Organization for Economic Coopera and Development in 1980. The E-Government Act of 2002 strives to enhance protection for personal information in government information systems or information collections by requiring that agencies conduct pr impact assessments (PIA). A PIA is an analysis of how personal information is collected, stored, shared, and managed in a federa l system. More specifically, according to Office of Management and Budget (OMB) guidance, a PIA is an analysis of how information ishandled. Specifically, a PIA is to (1) ensure that handling conforms to applicable legal, regulatory, and policy requirements regarding privacy; (2) determine the risks and effects of collecting, maintaining, and disseminating information in identifiable an electronic information system; and (3) examine and evaluate protections and alternative processes for handling information to mitigate potential privacy risks. Agencies must conduct PIAs (1) before developing or procuring information technology that collects, maintains, or disseminates information that is in a personally identifiable form; or (2) before initiating any new data collections involving personal information that will be collected, maintained, or disseminated using information technology if the same questions are asked of 10 or more people. To the extent that PIAs are made publicly available they provide explanations to the public about such things as the information that will be collected, why it is being collected, how to be used, and how the system and data will be maintained and protected. FISMA also addresses the protection of personal information. FISMA defines federal requirements for securing information a information systems that support federal agency operations and assets; it requires agencies to develop agencywide information security programs that extend to contractors and other provider federal data and systems. Under FISMA, information security means protecting information and information systems from unauthorized access, use, disclosure, disruption, modification destruction, including controls necessary to preserve authorized, or restrictions on access and disclosure to protect personal privacy, among other things. OMB is tasked with providing guidance to agencies on how to implement the provisions of the Privacy Act and the E-Government Act and has done so, beginning with guidance on the Privacy Act, issued in 1975. The guidance provides explanations for the various provisions of the law as well as detailed instructions for how to comply. OMB’s guidance on implementing the privacy provisions of the E-Government Act of 2002 identifies circumstances under which agencies must conduct PIAs and explains how to conduct them. OMB has also issued guidance on implementing the provisions of FISMA. Privacy Officers Have Gained Prominence at Several Federal Agencies While many agencies have had officials designated as focal points for privacy-related matters for some time, these positions have recently gained greater prominence at a number of agencies. A long- standing requirement has been in place for agency chief information officers to be responsible for implementing and enforcing privacy policies, procedures, standards, and guidelines, and for compliance with the Privacy Act. In 2004, we reported that of the 27 major agency chief information officers, 17 were responsible for privacy and 10 were not. In those 10 agencies, privacy was most often the responsibility of the Office of General Counsel and/or various offices focusing on compliance with the Freedom of Information Act and the Privacy Act. Steps have been taken recently to highlight the importance of privacy officers in federal agencies. For example, the Transportation, Treasury, Independent Agencies, and General Government Appropriations Act of 2005 required each agency covered by the act to have a chief privacy officer responsible for, among other things, “assuring that the use of technologies sustain, and do not erode, privacy protections relating to the use, collection, and disclosure of information in identifiable form.” Subsequently, in February 2005, OMB issued a memorandum to federal agencies requiring them to designate a senior official with overall agencywide responsibility for information privacy issues. This senior official was to have overall responsibility and accountability for ensuring the agency’s implementation of information privacy protections and play a central policy-making role in the agency’s development and evaluation of policy proposals relating to the agency’s collection, use, sharing, and disclosure of personal information. Prior to the OMB guidance, several agencies had already designated privacy officials at higher levels. The Internal Revenue Service had been one of the first, establishing its privacy advocate in 1993. In 2001, the Postal Service established a Chief Privacy Officer. More recently, as you know, Section 222 of the Homeland Security Act of 2002 had created the first statutorily required senior privacy official at any federal agency. This law mandated the appointment of a senior official at DHS to assume primary responsibility for privacy policy, including, among other things, assuring that the use of technologies sustains, and does not erode, privacy protections relating to the use, collection, and disclosure of personal information. Since being established, the DHS Privacy Office created a Data Privacy and Integrity Advisory Committee, made up of experts from the private and non-profit sectors and the academic community, to advise it on issues within DHS that affect individual privacy, as well as data integrity, interoperability, and other privacy- related issues. Through the Intelligence Reform Act in 2004, Congress expressed more broadly the sense that agencies with law enforcement or anti- terrorism functions should have a privacy and civil liberties officer. In keeping with that, Justice recently announced the appointment of a Chief Privacy and Civil Liberties Officer responsible for reviewing and overseeing the department’s privacy operations and complying with privacy laws. Justice has also announced plans to establish an internal Privacy and Civil Liberties Board made up of senior Justice officials to assist in ensuring that the department’s activities are carried out in a way that fully protects the privacy and civil liberties of Americans. Agency Privacy Officers Face a Number of Challenges The elevation of privacy officers at federal agencies reflects the growing demands that these individuals face in addressing privacy challenges on a day-to-day basis. Among these challenges, several that are prominent include (1) complying with the Privacy Act and the E-Government Act of 2002, (2) ensuring that data mining efforts do not compromise privacy protections, (3) controlling the collection and use of personal information obtained from commercial sources, and (4) addressing concerns about radio frequency identification technology. Complying with the Privacy Act and the E-Government Act of 2002 Although it has been on the books for more than 30 years, the Privacy Act of 1974 continues to pose challenges for federal agencies. In 2003, we reported that agencies generally did well with certain aspects of the Privacy Act’s requirements—such as issuing system-of-records notices when required—but did less well at other requirements, such as ensuring that information is complete, accurate, relevant, and timely before it is disclosed to a nonfederal organization. In discussing this uneven compliance, agency officials reported the need for additional OMB leadership and guidance to assist in difficult implementation issues in a rapidly changing environment. For example, officials had questions about the act’s applicability to electronic records. Additional issues included the low agency priority given to implementing the act and insufficient employee training on the act. These are all issues that chief privacy officers could be in a position to address. For example, working in concert with officials from OMB and other agencies, they are in a position to identify ambiguities in guidance and provide clarifications about the applicability of the Privacy Act. Further, the establishment of a chief privacy officer position and its relative seniority within an agency’s organizational structure could indicate that an agency places priority on implementing the act. Finally, a chief privacy officer could also serve as a champion for privacy awareness and education across an agency. The E-Government Act’s requirement that agencies conduct PIAs is relatively recent, and we have not yet made a comprehensive assessment of agencies’ implementation of this important provision. However, our previous work has highlighted challenges with respect to conduct of these assessments for certain applications. For example, in our work on federal agency use of information resellers, we found that few agency components reported developing PIAs for their systems or programs that make use of information reseller data. These agencies often did not conduct PIAs because officials did not believe they were required. Current OMB guidance on conducting PIAs is not always clear about when they should be conducted. We concluded that until PIAs are conducted more thoroughly and consistently, the public is likely to remain incompletely informed about the purposes and uses for the information agencies obtain from resellers. We recommended that OMB revise its guidance to clarify the applicability of the E-Gov Act’s PIA requirement (as well as Privacy Act requirements) to the use of personal information from resellers. Compliance with OMB’s PIA guidance was also an issue in our review of selected data mining efforts at federal agencies. In that review, although three of the five data mining efforts we assessed had conducted PIAs, none of these assessments fully complied with OMB guidance. Complete assessments are an important tool for agencies to identify areas of noncompliance with federal privacy laws, evaluate risks arising from electronic collection and maintenance of information about individuals, and evaluate protections or alternative processes needed to mitigate the risks identified. Agencies that do not take all the steps required to protect the privacy of personal information limit the ability of individuals to participate in decisions that affect them, as required by law, and risk the improper exposure or alteration of personal information. We recommended that the agencies responsible for the data mining efforts complete or revise PIAs as needed and make them available to the public. The DHS Privacy Office recently issued detailed guidance on conducting PIAs that may be helpful to departmental components as they develop and implement systems that involved personal information. The guidance notes that PIAs can be one of the most important instruments in establishing trust between the department and the public. As agencies develop or make changes to existing systems that collect personally identifiable information, it will continue to be critical for privacy officers to monitor agency activities and help ensure that PIAs are properly conducted so that their benefits can be realized. Ensuring that Data Mining Efforts Do Not Compromise Privacy Protections Many concerns have been raised about the potential for data mining programs at federal agencies to compromise personal privacy. In our May 2004 report on federal data mining efforts, we defined data mining as the application of database technology and techniques— such as statistical analysis and modeling—to uncover hidden patterns and subtle relationships in data and to infer rules that allow for the prediction of future results. We based this definition on the most commonly used terms found in a survey of the technical literature. As we noted in our report, mining government and private databases containing personal information raises a range of privacy concerns. In the government, data mining was initially used to detect financial fraud and abuse. However, its use has greatly expanded. Among other purposes, data mining has been used increasingly as a tool to help detect terrorist threats through the collection and analysis of public and private sector data. Through data mining, agencies can quickly and efficiently obtain information on individuals or groups by exploiting large databases containing personal information aggregated from public and private records. Information can be developed about a specific individual or a group of individuals whose behavior or characteristics fit a specific pattern. The ease with which organizations can use automated systems to gather and analyze large amounts of previously isolated information raises concerns about the impact on personal privacy. Before data aggregation and data mining came into use, personal information contained in paper records stored at widely dispersed locations, such as courthouses or other government offices, was relatively difficult to gather and analyze. In August 2005, we reported on five different data mining efforts at selected federal agencies, noting that although the agencies responsible for these data mining efforts took many of the steps needed to protect the privacy and security of personal information used in the efforts, none followed all key procedures. Most of the agencies provided a general public notice about the collection and use of the personal information used in their data mining efforts. However, fewer followed other required steps, such as notifying individuals about the intended uses of their personal information when it was collected or ensuring the security and accuracy of the information used in their data mining efforts. In addition, as I previously mentioned, although three of the five agencies completed privacy impact assessments of their data mining efforts, none fully complied with OMB guidance. We made recommendations to the agencies responsible for the five data mining efforts to ensure that their efforts included adequate privacy and security protections. In March 2004, an advisory committee chartered by the Department of Defense issued a comprehensive report on privacy concerns regarding data mining in the fight against terrorism. The report made numerous recommendations to better ensure that privacy requirements are clear and stressed that proper oversight be in place when agencies engage in data mining that could include personal information. Agency privacy offices can provide a degree of internal oversight to help ensure that privacy is fully addressed in agency data mining activities. Controlling the Collection and Use of Personal Information Obtained from Commercial Sources Recent security breaches at large information resellers, such as ChoicePoint and LexisNexis, have highlighted the extent to which such companies collect and disseminate personal information. Information resellers are companies that collect information, including personal information about consumers, from a wide variety of sources for the purpose of reselling such information to their customers, which include both private-sector businesses and government agencies. Before advanced computerized techniques made aggregating and disseminating such information relatively easy, much personal information was less accessible, being stored in paper-based public records at courthouses and other government offices or in the files of nonpublic businesses. However, information resellers have now amassed extensive amounts of personal information about large numbers of Americans, and federal agencies access this information for a variety of reasons. A major task confronting federal agencies, especially those engaged in antiterrorism tasks, has been to ensure that information obtained from resellers is being appropriately used and protected. To this end, in September 2005, the DHS Privacy Office held a public workshop to examine the policy, legal, and technology issues associated with the government’s use of reseller data for homeland security. Participants provided suggestions on how the government can ensure that privacy is protected while enabling the agencies to analyze reseller data. We recently testified before this subcommittee on critical issues surrounding the federal government’s acquisition and use of personal information from information resellers. In our review of the acquisition of personal information from resellers by DHS, Justice, the Department of State, and the Social Security Administration, agency practices for handling this information did not always reflect the Fair Information Practices. For example, although agencies issued public notices on information collections, these did not always notify the public that information resellers were among the sources to be used, a practice inconsistent with the principle that individuals should be informed about privacy policies and the collection of information. And again, a contributing factor was ambiguities in guidance from OMB regarding the applicability of privacy requirements in this situation. As I mentioned previously, we recommended that OMB revise its guidance to clarify the applicability of governing laws—both the Privacy Act and the E-Gov Act—to the use of personal information from resellers. In July 2005, we reported on shortcomings at DHS’s Transportation Security Administration (TSA) in connection with its test of the use of reseller data for the Secure Flight airline passenger screening program. TSA did not fully disclose to the public its use of personal information in its fall 2004 privacy notices, as required by the Privacy Act. In particular, the public was not made fully aware of, nor had the opportunity to comment on, TSA’s use of personal information drawn from commercial sources to test aspects of the Secure Flight program. In September 2004 and November 2004, TSA issued privacy notices in the Federal Register that included descriptions of how such information would be used. However, these notices did not fully inform the public before testing began about the procedures that TSA and its contractors would follow for collecting, using, and storing commercial data. In addition, the scope of the data used during commercial data testing was not fully disclosed in the notices. Specifically, a TSA contractor, acting on behalf of the agency, collected more than 100 million commercial data records containing personal information such as name, date of birth, and telephone number without informing the public. As a result of TSA’s actions, the public did not receive the full protections of the Privacy Act. In its comments on our findings, DHS stated that it recognized the merits of the issues we raised, and that TSA acted immediately to address them. In our report on information resellers, we recommended that the Director, OMB, revise privacy guidance to clarify the applicability of requirements for public notices and privacy impact assessments to agency use of personal information from resellers and direct agencies to review their uses of such information to ensure it is explicitly referenced in privacy notices and assessments. Further, we recommended that agencies develop specific policies for the use of personal information from resellers. Until privacy requirements are better defined and broadly understood, agency privacy officers are likely to continue to face challenges in helping ensure that their agencies are providing appropriate privacy protections. Addressing Concerns about Radio Frequency Identification Technology Specific issues about the design and content of identity cards also raise broader privacy concerns associated with the adoption of new technologies such as radio frequency identification (RFID). RFID is an automated data-capture technology that can be used to electronically identify, track, and store information contained on a tag. The tag can be attached to or embedded in the object to be identified, such as a product, case, or pallet. RFID technology provides identification and tracking capabilities by using wireless communication to transmit data. In May 2005, we reported that major initiatives at federal agencies that use or propose to use the technology included physical access controls and tracking assets, documents, or materials. For example, DHS was using RFID to track and identify assets, weapons, and baggage on flights. The Department of Defense was also using it to track shipments. In our May 2005 report we identified several privacy issues related to both commercial and federal use of RFID technology. Among these privacy issues are notifying individuals of the existence or use of the technology; tracking an individual’s movements; profiling an individual’s habits, tastes, or predilections; and allowing for secondary uses of information. The extent and nature of the privacy issues depends on the specific proposed use. For example, using the technology for generic inventory control would not likely generate substantial privacy concerns. However, the use of RFIDs by the federal government to track the movement of individuals traveling within the United States could generate concern by the affected parties. A number of specific privacy issues can arise from RFID use. For example, individuals may not be aware that the technology is being used and that it could be embedded in items they are carrying and thus used to track them. Three agencies indicated to us that employing the technology would allow for the tracking of employees’ movements. Tracking is real-time or near-real-time surveillance in which a person’s movements are followed through RFID scanning. Media reports have described concerns about ways in which anonymity is likely to be undermined by surveillance. Further, public surveys have identified a distinct unease with the potential ability of the federal government to monitor individuals’ movements and transactions. Like tracking, profiling—the reconstruction of a person’s movements or transactions over a specific period of time, usually to ascertain something about the individual’s habits, tastes, or predilections—could also be undertaken through the use of RFID technology. Because tags can contain unique identifiers, once a tagged item is associated with a particular individual, personally identifiable information can be obtained and then aggregated to develop a profile of the individual. Both tracking and profiling can compromise an individual’s privacy and anonymity. Concerns also have been raised that organizations could develop secondary uses for the information gleaned through RFID technology; this has been referred to as “mission-” or “function- creep.” The history of the Social Security number, for example, gives ample evidence of how an identifier developed for one specific use has become a mainstay of identification for many other purposes, governmental and nongovernmental. Secondary uses of the Social Security number have been a matter not of technical controls but rather of changing policy and administrative priorities. As agencies take advantage of the benefits of RFID technology and implement it more widely, it will be critical for privacy officers to help ensure that a full consideration is made of potential privacy issues, both short-term and long-term, as the technology is implemented. In summary, privacy officers at federal agencies face a range of challenges in working to ensure that individual privacy is protected, and today I have discussed several of them. It is clear that advances in technology can present both opportunities for greater agency efficiency and effectiveness as well as the danger, if unaddressed, of eroding important privacy protections. Technological advances also mean there is a need to keep governmentwide privacy guidance up- to-date, and agency privacy officers will depend on OMB for leadership in this area. Even without a consideration of technological evolution, privacy officers need to be vigilant to ensure that agency officials are continually mindful of their privacy responsibilities. Fortunately, tools are available—including the requirements for PIAs and Privacy Act public notices—that can help ensure that the right operational decisions are made about the acquisition, use, and storage of personal information. By using these tools effectively, agencies have the opportunity to gain greater public confidence that their actions are in the best interests of all Americans. Mr. Chairman, this concludes my testimony today. I would happy to answer any questions you or other members of the subcommittee may have. Contacts and Acknowledgements If you have any questions concerning this testimony, please contact Linda Koontz, Director, Information Management, at (202) 512-6240, or koontzl@gao.gov. Other individuals who made key contributions include Barbara Collier, John de Ferrari, David Plocher, and Jamie Pressman. Attachment I: Selected GAO Products Related to Privacy Issues Personal Information: Agencies and Resellers Vary in Providing Privacy Protections. GAO-06-609T. Washington, D.C.: April 4, 2006. Personal Information: Agency and Reseller Adherence to Key Privacy Principles. GAO-06-421. Washington, D.C.: April 4, 2006. Data Mining: Agencies Have Taken Key Steps to Protect Privacy in Selected Efforts, but Significant Compliance Issues Remain. GAO- 05-866. Washington, D.C.: August 15, 2005. Aviation Security: Transportation Security Administration Did Not Fully Disclose Uses of Personal Information during Secure Flight Program Testing in Initial Privacy Notices, but Has Recently Taken Steps to More Fully Inform the Public. GAO-05- 864R. Washington, D.C.: July 22, 2005. Identity Theft: Some Outreach Efforts to Promote Awareness of New Consumer Rights are Under Way. GAO-05-710. Washington, D.C.: June 30, 2005. Information Security: Radio Frequency Identification Technology in the Federal Government. GAO-05-551. Washington, D.C.: May 27, 2005. Aviation Security: Secure Flight Development and Testing Under Way, but Risks Should Be Managed as System is Further Developed. GAO-05-356. Washington, D.C.: March 28, 2005. Electronic Government: Federal Agencies Have Made Progress Implementing the E-Government Act of 2002. GAO-05-12. Washington, D.C.: December 10, 2004. Social Security Numbers: Governments Could Do More to Reduce Display in Public Records and on Identity Cards. GAO-05-59. Washington, D.C.: November 9, 2004. Federal Chief Information Officers: Responsibilities, Reporting Relationships, Tenure, and Challenges, GAO-04-823. Washington, D.C.: July 21, 2004. Data Mining: Federal Efforts Cover a Wide Range of Uses, GAO-04- 548. Washington, D.C.: May 4, 2004. Aviation Security: Computer-Assisted Passenger Prescreening System Faces Significant Implementation Challenges. GAO-04-385. Washington, D.C.: February 12, 2004. Privacy Act: OMB Leadership Needed to Improve Agency Compliance. GAO-03-304. Washington, D.C.: June 30, 2003. Data Mining: Results and Challenges for Government Programs, Audits, and Investigations. GAO-03-591T. Washington, D.C.: March 25, 2003. Technology Assessment: Using Biometrics for Border Security. GAO-03-174. Washington, D.C.: November 15, 2002. Information Management: Selected Agencies’ Handling of Personal Information. GAO-02-1058. Washington, D.C.: September 30, 2002. Identity Theft: Greater Awareness and Use of Existing Data Are Needed. GAO-02-766. Washington, D.C.: June 28, 2002. Social Security Numbers: Government Benefits from SSN Use but Could Provide Better Safeguards. GAO-02-352. Washington, D.C.: May 31, 2002. Attachment 2: The Fair Information Practices The Fair Information Practices are not precise legal requirements. Rather, they provide a framework of principles for balancing the need for privacy with other public policy interests, such as national security, law enforcement, and administrative efficiency. Ways to strike that balance vary among countries and according to the type of information under consideration. The version of the Fair Information Practices shown in table 1 was issued by the Organization for Economic Cooperation and Development (OECD) in 1980 and has been widely adopted. | Summary: Advances in information technology make it easier than ever for the federal government to obtain and process personal information about citizens and residents in many ways and for many purposes. To ensure that the privacy rights of individuals are respected, this information must be properly protected in accordance with current law, particularly the Privacy Act and the E-Government Act of 2002. These laws prescribe specific activities that agencies must perform to protect privacy, and the Office of Management and Budget (OMB) has developed guidance on how and in what circumstances agencies are to carry out these activities. Many agencies designate officials as focal points for privacy-related matters, and increasingly, many have created senior positions, such as chief privacy officer, to assume primary responsibility for privacy policy, as well as dedicated privacy offices. GAO was asked to testify on key challenges facing agency privacy officers. To address this issue, GAO identified and summarized issues raised in its previous reports on privacy. Agencies and their privacy officers face growing demands in addressing privacy challenges. For example, as GAO reported in 2003, agency compliance with Privacy Act requirements was uneven, owing to ambiguities in guidance, lack of awareness, and lack of priority. While agencies generally did well with certain aspects of the Privacy Act's requirements--such as issuing notices concerning certain systems containing collections of personal information--they did less well at others, such as ensuring that information is complete, accurate, relevant, and timely before it is disclosed to a nonfederal organization. In addition, the E-Gov Act requires that agencies perform privacy impact assessments (PIA) on such information collections. Such assessments are important to ensure, among other things, that information is handled in a way that conforms to privacy requirements. However, in work on commercial data resellers, GAO determined in 2006 that many agencies did not perform PIAs on systems that used reseller information, believing that these were not required. In addition, in public notices on these systems, agencies did not always reveal that information resellers were among the sources to be used. To address such challenges, chief privacy officers can work with officials from OMB and other agencies to identify ambiguities and provide clarifications about the applicability of privacy provisions, such as in situations involving the use of reseller information. In addition, as senior officials, they can increase agency awareness and raise the priority of privacy issues. Agencies and privacy officers will also face the challenge of ensuring that privacy protections are not compromised by advances in technology. For example, federal agency use of data mining--the analysis of large amounts of data to uncover hidden patterns and relationships--was initially aimed at detecting financial fraud and abuse. Increasingly, however, the use of this tool has expanded to include purposes such as detecting terrorist threats. GAO found in 2005 that agencies employing data mining took many steps needed to protect privacy (such as issuing public notices), but none followed all key procedures (such as including in these notices the intended uses of personal information). Another new technology development presenting privacy challenges is radio frequency identification (RFID), which uses wireless communication to transmit data and thus electronically identify, track, and store information on tags attached to or embedded in objects. GAO reported in 2005 that federal agencies use or propose to use the technology for physical access controls and tracking assets, documents, or materials. For example, the Department of Defense was using RFID to track shipments. Although such applications are not likely to generate privacy concerns, others could, such as the use of RFIDs by the federal government to track the movement of individuals traveling within the United States. Agency privacy offices can serve as a key mechanism for ensuring that privacy is fully addressed in agency approaches to new technologies such as data mining and RFID. | 6,231 | 803 | gov_report | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATION [0001] This application is a divisional of U.S. application Ser. No. 14/281,879 filed on 19 May 2014, which further claims the benefit of U.S. Provisional Application No. 61/824,365 filed on 17 May 2013, the contents of which are incorporated herein by reference. A claim of priority is made. FIELD OF THE INVENTION [0002] The present invention relates to an apparatus for harvesting sugar crops. More particularly, the present invention relates to an apparatus for harvesting sweet sorghum in a form required for cold weather storage of sugars in commercially useful piles. BACKGROUND OF THE INVENTION [0003] Nations seek to produce biofuels and biochemicals from farm crops for economic, environmental and national security reasons. Sugar cane is currently the world's lowest-cost raw material for the production of biomass energy and biochemicals. The crop can produce high yields of sugar and fiber at relatively low agricultural costs. However, sugar cane cannot be rotated with food crops or even grown in many areas of the world, including those with degraded, infertile land or arid lands with low rainfall or with short growing and harvesting seasons (less than 180 days). Nations want to use these lands in way that doesn't compete with food crops. [0004] Many nations could potentially use a similar, but lower input, cane sugar crop called sweet sorghum, which can grow on arid, saline and alkaline soils in about 120 days. Compared to sugar cane, sweet sorghum can produce nearly as much sugar and fiber with lower agricultural costs, including seeds, nitrogen fertilizer, water and land. However, pest problems can make it difficult to grow sweet sorghum in southern areas with long growing and harvesting seasons. Generally pests don't pose significant problems in colder, northern regions. [0005] The overall problem in colder, northern regions is the production of various products from sweet sorghum is not cost-competitive when the crop must be hauled to the plant and processed only during the short harvest season. The solution is to delay deterioration of the sugars by storing the crop as a de-leafed whole plant in the cold weather near local farms to extend the hauling and process season over the winter (like accomplished by the sugar beet industry). [0006] The storage of sugar crops depends on providing conditions that allow the crop to live at low metabolism. Sugar losses are caused by normal respiration as the stalks use sugar as an energy source at above freezing temperatures. Sugar crops respire and deteriorate more rapidly under several conditions, including: when damaged by disease or insects during growth; when burned; if the crop is cut or bruised during mechanical harvesting; if the crop is subjected to elevated temperatures above freezing; or by an accumulation of environmental or climatic effects including heat, sunlight and alternating frosts and thaws. [0007] As practiced on a commercial scale by the sugar beet industry, storage of sugars within sugar beets outside during cold weather requires harvest as a whole plant with minimal damage in order to slow deterioration over the winter. In addition, leaf removal is also likely necessary to allow circulation of air within large commercial piles of the crop to prevent over-heating from excessive fermentation. Piles are typically sealed on the outside with hay or plastic. Poor ventilation or desiccation reduces the oxygen content in piles causing fermentation, decay and sugar loss. For successful commercial storage of sweet sorghum sugars, a harvesting system must be able to strip the leaves with minimal stalk damage. [0008] The large surface area of the sweet sorghum leaves means pile ventilation will be blocked. Moreover, it would be preferable to reject most of the leaves back onto the land as soil conditioner and also to get them out of the way for piling and later milling. But the storage methodology practiced by the sugar beet industry, which requires removal of virtually all leaf matter back to the soil, cannot be assumed to be most effective for sweet sorghum. Stalks pile with less density and greater amount of air spaces than beets. It is unknown whether some leaves should be used to slow the air flow or if bigger piles are needed with better sealing when temperatures are cold and about to warm. At least initially, a wide range of leaf removal is preferable until sufficient data can be obtained through testing of large piles under actual variable weather conditions to determine how much leaf removal is optimal for commercial storage of sweet sorghum. [0009] Sugar loss increases with cutting. N. J. King in Producers' Review 1972, 62, (7), 66-67 reported for sugar cane: “While full-length (6-ft) cane stalks with two cut ends dry out rapidly, the same stalks, when passed through a chopper harvester, will be cut into six 12 inch billets having a total of 12 ends which do not dry rapidly because of the protection afforded by surrounding billets. The result is faster deterioration than in the case of the 6-ft stalks, the rate increasing with reduction in billet size, short billets also splitting more easily than long ones and thus being susceptible to attacks by bacteria.” [0010] Unfortunately, commercial harvesting systems for sweet sorghum are not currently available that can strip the leaves from stalks with minimal damage. Sweet sorghum can be harvested, handled and processed with equipment used for sugar cane (since both are similarly-shaped cane sugar crops). But commercial sugar cane harvesters, including both choppers and soldiers, are also not designed to strip the leaves with minimal damage even though it would be advantageous. Nor is there commercial equipment available for either pre-harvest or post-harvest that could strip the leaves from stalks of either sweet sorghum or sugar cane with minimal damage. [0011] Commercial chopper (billet) harvesters are used by most commercial sugar cane operations and are also proposed by most sweet sorghum projects, especially those in warmer southern climates. Sugar cane chopper harvesters can strip the leaves to the farm field, handle fairly narrow rows and recover lodged stalks, but the chopping into billets damages the stalks far too much for long-term storage in colder northern climates. [0012] Commercial soldier (whole stalk) harvesters can harvest sugar cane stalks with little damage, even though the chains on which the stalks ride may cause excessive damage. But commercial soldier harvesters are being phased out for use with sugar cane and also have little use for sweet sorghum, because these whole stalk harvesters cannot strip the leaves, and thus require burning of the leaves in the field, which may be prohibited in some areas. Burning also causes infections and loss of sugars. Moreover, crop yields cannot be maximized because the harvesters cannot handle narrow row spacing or harvest lodged stalks fallen by heavy crop weights. [0013] Some researchers have proposed pre-harvest leaf removal using high-boys, driven above the crop with the wheels between the rows, and equipped with circulating rubber finger strippers that beat the leaves off the stalks from both sugar cane and sweet sorghum stalks standing in the field. But these machines cannot remove leaves from lodged stalks and appear to considerably damage the stalks; while the rubber strippers wear out too quickly for commercial use. Some commercial sugar cane operations, especially in Cuba, use stationary sugar cane cleaning tables that can strip leaves, but this requires cutting, and thus damaging, the stalks. [0014] A few sugar cane prototype harvesters have been built to harvest whole stalks of sugar cane with minimal damage while stripping the leaves. Such harvesters also tend to be designed for the stalks to ride on a conveyor, instead of chains, to minimize stalk damage and also to harvest narrow rows and lodged stalks. But these prototype sugar cane harvesters have not been fully developed for commercial harvesting of sugar cane, and until now, have not been modified for, or tested on, sweet sorghum. [0015] Besides the essential need to strip the leaves with minimal damage, modification of sugar cane harvesters for use with sweet sorghum must also address some crop differences. For example, sweet sorghums often have more numerous, thinner and softer stalks, less numerous and shorter leaves, and grain heads, compared to sugar cane. The present invention provides a solution to these needs and other problems, and offers other advantages over the prior art. [0016] Research has indicated that very low sugar losses may be had within one meter high piles of de-leafed and whole sweet sorghum stalks stored outdoors on a farm field in southern Minnesota during the winter. In order to provide for optimal storage and processing conditions with the least amount of sugar deterioration, sorghum should be de-leafed in the field, with minimal damage to the stalk, prior to transport to processing plants. This invention solves this, and other, problems and provides advantages over the prior art. [0017] The specific problem addressed by these inventive changes is the lack of a harvester that can economically deliver sweet sorghum as de-leafed and undamaged whole stalks required for storage of the crop in climates with cold winters, while also meeting other requirements including the minimization of manual labor through economy-of-scale and mechanization, and also yield maximization through the harvesting of narrow rows (30 to 40 inches), cutting the stalks at ground level and recovering fallen stalks. [0018] The proposed solution is to modify a harvester that has been designed to meet these requirements for the somewhat similar crop sugar cane (whose sugars can also be stored albeit for shorter periods in warmer climates), and these modifications represent the novelty of the invention. BRIEF SUMMARY OF THE INVENTION [0019] The apparatus disclosed herein solves the significant problems related to harvesting that currently prevent the commercialization of the agricultural crop sweet sorghum in northern regions. The presently described harvester apparatus can reduce or substantially eliminate at least some of the following, for example: [0020] (1) the problem of sugar deterioration during storage is slowed by harvesting whole stalks with minimal damage; [0021] (2) the problem of increased sugar deterioration during harvesting and storage is slowed by preventing damage from shattering when cutting the stalks at ground level; [0022] (3) the problem of increased sugar deterioration in large commercial storage piles is slowed by using the harvester to strip (clean) the leaves and blow them back onto the farm land (without burning); [0023] (4) the problem of lower food yields on crop land is reduced by topping and collecting the grain-heads for feed by-product; [0024] (5) the problem of reduced crop yields during planting is solved by harvesting rows as narrow as 30 inches (compared to wider spacing for sugar cane); [0025] (6) the problem of crop yield loss during harvesting is solved by cutting the stalks at ground level (noting that the largest diameter and yield of the stalks is at the bottom of the stalks), recovering over 95% of the stalks, collecting at least 90 percent of lodged cane and pile the cane for loading of at least 98% of the cane; [0026] (7) the problem of uneconomical harvesting is solved by achieving harvest rates of 30 to 100 tons per hour, and capital costs, fuel and maintenance should be comparable to chopper harvesters; and [0027] (8) the problem of manual labor is solved by totally mechanized harvesting. [0028] The design specifications proposed for sweet sorghum harvesters were developed and demonstrated to design and build specific equipment. Development was based on modification of technology established with prototype and commercial sugar cane harvesters, with special consideration for the need to harvest the crop in the form required for cold weather storage of sugars in commercial piles like that practiced with sugar beets. Harvesters used for sugar cane can best serve as the design basis since both crops are tall, high-yielding, leafy cane crops with a tendency to lodge and have similar uses. It is important to use experiences gained with sugar cane since it has proved to be the most difficult crop to machine harvest. [0029] A sweet sorghum harvester may be designed and built by selecting and modifying a prototype whole stalk sugar cane harvester, such as the popular Centurion harvester, or a chopper (billet) harvester such as the Ausoft. The selection may be based on the harvester's design to harvests whole stalks with minimal damage while stripping the leaves, harvesting narrow rows, recovering 96% of the stalks, collecting lodged cane, and achieving rates of 30 tons per hour with total mechanization. [0030] The elemental designs are intended to meet product specifications required to solve the problems related to the harvesting of sweet sorghum in northern regions. The sugar deterioration problem caused by shattering when cutting the stalks at ground level is solved by novel base cutters. The problem of sugar deterioration during storage caused by the blockage of pile ventilation by leaves is solved by a leaf stripping mechanism. The reduced food yield problem, caused by the lack of a collection system for the topped grainheads, is solved by dividing the bin for collection. Other miscellaneous harvesting problems caused by the use of a harvester for sweet sorghum that is set for sugar cane are solved by various simple modifications as described below. [0031] Additional advantages and features of the invention will be set forth in part in the description which follows, and in part, will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. BRIEF DESCRIPTION OF THE DRAWINGS [0032] FIG. 1 illustrates a prototype whole-stalk sugar cane harvester. [0033] FIG. 2 provides another view of a whole stalk harvester showing additional details. [0034] FIG. 3 illustrates crop dividers which may be located at the front end of a harvester. [0035] FIG. 4 illustrates an exemplary harvester with inclined base cutters. [0036] FIGS. 5 a and 5 b illustrate how inclined base cutter discs the stalk at the base. [0037] FIGS. 6 a and 6 b illustrate a general arrangement of pivots for base cutter gearboxes. [0038] FIG. 7 illustrates a restraining arrangement to set the angle of the pivot vertical shaft. [0039] FIG. 8 illustrates another view of vertical shaft with spring loading device. [0040] FIG. 9 illustrates notched cutter blades with exemplary dimensions. [0041] FIG. 10 is a diagram of tire-like structures which grip the cut stalk and push it to the rear. [0042] FIG. 11 shows the pivoted mounting of a blade cutter gearbox. [0043] FIG. 12 shows the vertical axis of the gearbox. [0044] FIG. 13 shows another view of the gearbox. [0045] FIG. 14 illustrates the blade discs mounted to a vertical shaft. [0046] FIG. 15 diagrams the base cutter discs. [0047] FIG. 16 illustrates the rubber cylinder used to secure the blades on the base cutter disc. [0048] FIG. 17 illustrates a possible restraining arrangement for the base cutter vertical shaft. [0049] FIG. 18 illustrates an alternative restraining arrangement. [0050] FIG. 19 shows one method of spring loading the base cutter vertical shafts. [0051] FIG. 20 shows an alternative mechanism for spring loading the base cutter using adjustable springs. [0052] FIG. 21 depicts a set of notched cutter blades. [0053] FIG. 22 illustrates the blade mounting sockets on disc cutter plates. [0054] FIG. 23 shows shaft drums with guard plates. [0055] FIG. 24 depicts the tire structures mounted to the base cutter discs and used to pull the stalk rearward. [0056] FIG. 25 is a closer view of the tire structures. [0057] FIG. 26 shows the holes drilled into the tire to make it more resilient. [0058] FIG. 27 illustrates the feed conveyor, brushes and feed roll. [0059] FIG. 28 is a drawing showing an arrangement of base cutter mechanism and the associated apparatus which move stalks through the system. [0060] FIG. 29 depicts a side view of a base cutter and nip arrangement. [0061] FIG. 30 illustrates another view of the conveyor and cutter systems with fan and cowling. [0062] FIG. 31 is a photograph of the brush elements. [0063] FIG. 32 shows the two-roller nip to which the conveyor feeds stalks. [0064] FIG. 33 illustrates an extractor fan. [0065] FIG. 34 shows an alternative arrangement where no nip rollers are placed downstream of the brushes. [0066] FIG. 35 depicts a lower hurler roller. [0067] FIG. 36 depicts an upper hurler roller. [0068] FIG. 37 illustrates the soft rubber wheels of the upper hurler. [0069] FIG. 38 illustrates the lower hurler with hard rubber tires. [0070] FIG. 39 shows a reworked chain case used to cover the hurler drive. [0071] FIG. 40 shows a top hurler pivot and lubrication system. [0072] FIG. 41 shows the debridging flail, stabilizing clamps and side breakers. [0073] FIG. 42 depicts an air blast deflector. [0074] FIG. 43 depicts a hydraulic drive of the fan system. [0075] FIG. 44 shows the cover for a top brush. [0076] FIG. 45 illustrates the entry of an extractor fan. [0077] FIG. 46 shows a rubber belting cover linking the top brush area to the fan entry. [0078] FIG. 47 depicts a fan outlet. [0079] FIG. 48 shows belting attached to a fan. [0080] FIG. 49 depicts a front axle support. [0081] FIG. 50 depicts a rear axle support. [0082] FIG. 51 depicts an axle support system in place. [0083] FIG. 52 illustrates a topper unit. [0084] FIG. 53 shows another view of a topper structure. [0085] FIG. 54 shows a bin frame. [0086] FIG. 55 depicts the telescope section of a bin before installation. [0087] FIG. 56 depicts bin sidings. [0088] FIG. 57 shows a bin structure in context. [0089] FIG. 58 depicts rubber sheeting that may be used to separate a bin into sections. [0090] FIG. 59 illustrates a cushion used to support bin doors. [0091] FIG. 60 a shows the catch-rubber and FIG. 60 b shows the weight that keeps the catch-rubber forward. [0092] FIG. 61 shows extensions to the doors for the special front compartment. [0093] FIG. 62 shows mounted cross-tube in the main axel beam area. DETAILED DESCRIPTION [0094] The harvester disclosed herein may be based on a whole stalk harvester apparatus, a chopper harvester apparatus, or the like. The elements of the design modifications are discussed in terms of modifications to a whole stalk harvester, but are equally applicable to the chopper type as well. The design of the various elements often requires delicate balancing; they are inextricably intertwined and often opposing. The modifications discussed below represent components developed for a preferred embodiment of a sweet sorghum harvester. [0095] FIGS. 1 and 2 illustrate a prototype whole-stalk sugar cane harvester designed to harvest whole stalks of sugar cane with minimal damage while stripping the leaves, handling narrow rows and harvesting even lodged stalks. Referring to FIG. 1, dividers 102 at the front of the harvester gather cane in the row and separate it from cane in adjoining rows. The bin 104 in the rear accumulates and discharges cane into piles. FIG. 2 shows additional detail of harvester components. Base-cutters 202 sever the cane at ground level. A feed conveyor 204 carries the cane through the machine. A de-trashing roll 206 removes free trash including leaves. Hurler drums 208 pull cane from the conveyor and throw it into the bin. Rubber comb fingers 210 catch as many of the leaves as possible. A flail rotor 214 ejects trash caught by comb fingers and an air blast 212. The air blast 212, directed against cane flow and downwards towards the flail, separates trash from cane. A topper 216, including a pair of sharp-toothed discs rotating outwards against ledger plates, severs the tops of the cane to length. [0096] As shown in FIG. 2, the harvester cuts 202 the cane at the base but improvements are always sought to reduce machine wear and extraneous matter. The design of the conveyor 204 requires finding the right compromise between an aggressive feed, which damages cane, and a kinder feed, which can lead to chokes in the throat. The hurler drum 208 requires the right combination of resilience and wear resistance while making replacement as cheap as possible. The harvester's topping device 216 is optimized for clean removal of seed head tops, while minimizing stalk losses and damage. The harvester bin 104 is optimized to neatly accumulate an optimal-size bundle of stalks for loading, dump the bundles in neat piles onto the ground (clear of trash) for collection by the loaders, and discharge the bundles away from the machine and the path of the machine during subsequent passes on the field. Further reductions in extraneous matter are expected to come mainly from improvements in trash scrape and loading methods. [0097] In an exemplary embodiment, the production rate of the machine might achieve an objective pour rate of 0.75 to 1 tons per minute in green cane (or a forward speed of 3.75 kilometers per hour in 80 ton per hectare stalks planted at 1.5 meter row spacing). A harvester may be fitted with an engine providing 165 horsepower (123 kilowatts) gross output. A harvester engine may drive a 3-outlet splitter box with connected hydro-pump, steering pump and two tandem gear pumps. The hydraulic drive, hydrostatic vehicle transmission, 4-wheel drive and variable displacement motors may be equipped with high and low speed ranges. An exemplary harvester may have 15 hydraulic motors of the orbital type including those for cleaning and oil cooler fans. The consumption of power is mainly by the various harvesting operations including the base cutter and dividers, but also for traction, the cleaning fan and engine auxiliaries. In addition, there are pump, circuit, motor and other losses. Moreover, the motors should have ample back-up torque to cope with sudden overloads. [0098] A harvester further optimized for field performance and stability has rear-steer configuration to get maximum maneuverability and satisfactory axle load distribution for the long length needed to handle whole-stalks of cane going through it horizontally. A wheelbase of dimensions similar to a tractor (approximately 2.3 meters), provides a tight turning circle for headlands. A base cutter and divider can be lifted up high at the end of a row, to give a 20 degree clearance angle. There may be even more clearance at the back, when the bin doors have been raised. In an exemplary embodiment, the working limit for side-slope is 15 degrees. [0099] A preferred embodiment of a harvester modified to optimize the conditions of harvested stalk for processing includes at least one of the following harvester components: [0100] Dividers, [0101] Base-cutters, [0102] Feed conveyor, [0103] De-thrashing roll, [0104] Brush rolls, [0105] Hurler drums, [0106] De-bridging thrash roller, [0107] Air blast, [0108] Toppers, or [0109] Bin. Each will be discussed in turn. Dividers [0110] Two dividers may be located at the front of the harvester 102 ( FIGS. 1 and 3 ). Dividers are used to capture the stalks to be harvested and deflect to the side anything outside the capture area. Modification is usually needed because the dividers used for sugar cane harvesters have been designed for harvesting rows of one to two meters. In order to allow for narrow rows and higher yields, an optimal set of dividers may be spaced to accept dual crop rows of at most 30 inches apart. Base Cutters [0111] Base cutters sever the stalks at ground level and lift them into the throat. The stalks of relatively large-diameter and fibrous crops like sugarcane, sorghum, and coppiced trees are already cut mechanically with rotating discs onto which sharp-edged blades are attached. [0112] Both single disc cutters and twin (counter-rotating) disc-cutters are in use, typically spinning with a blade-tip speed of 3-6,000 ft per minute. A variant of the disc cutter is to have notches cut in the rim of a circular disc and not to use detachable blades. [0113] If the cutting device is forming the front of a “combine” sugarcane harvester, the counter-rotating discs also act as the gathering mechanism, pushing the cut stalks rearwards into a conveying mechanism. To make this rearward movement more positive it is typical to have one or more horizontal rollers over the twin discs. [0114] If the discs are running parallel to the ground, the disc, and structures on the disc that hold the detachable blades, experience considerable friction. FIG. 4 illustrates how twin disc cutters may be run at an appropriate angle to the ground to minimize power consumption in a typical sugar cane harvester. To reduce the consequent power consumption of these cutting devices to acceptable levels, they may be run in an inclined manner 400. The horizontal feed roller is situated over the base cutter 402. [0115] FIGS. 5 a and 5 b illustrate a front view of inclined base cutter discs and the cusp-shaped area consequential on their inclined plane. The cutters cannot cut the stalks in the center of the row close to the ground unless the crop is growing on a ridge. This inclination leaves a cusp-shaped area 500 in the center of the row where stalks are cut appreciably higher than desired. Traditionally the crop is grown on a low ridge 502 ( FIG. 5 b ) to fit the cusp-shaped area as well as possible. In many circumstances there are other advantages to growing the crop in this manner, but sometimes it is neither desirable, or possible, to do so and the crop may be growing from level soil. The modifications disclosed herein address this problem by inclining the shafts of the spinning discs in the plane viewed from the front of the machine, to reduce the missed area by the cusp when the whole mechanism is also inclined forward. Cane cutters may address the problem using pivoted, ground-following blades. This embodiment addresses the problem using rigidly mounted spinning disc cutters. [0116] Evidence indicates that the best way of handling the stalks is with a continuous notched disc, similar to a circular saw. The re-growth of the stubble, wherever that is appropriate, is superior after the saw-type action. Unfortunately stones can readily damage the notched discs and a large circular saw blade is very expensive. A simple notched disc may not be sufficiently aggressive. This matter is addressed with a blade shape and mounting which secures a saw-like action that uses replaceable sections. To further protect the blades an apparatus is proposed of reducing stone damage, as explained below. [0117] The counter-rotating disks of the traditional base cutter form a nip into which stones and other uncuttable objects can be taken and exert great forces on the structure and drives for the discs, as well as damage to the sharp blades. If the drive shafts to the discs are pivoted in such a way that they can easily move apart in response to excessive forces in the nip, the blades and mechanism generally will be protected from damage. This allows a lighter construction and a great increase in the longevity of the sharp blades. [0118] The horizontally-rotating rollers that help feed the crop across the blades into the conveying mechanism of conventional cane harvesters are fairly complicated to install and may damage the crop. Equipping the twin discs with resilient pads to provide additional rearward moving force to the crop, the rollers would not be necessary. This would save costs, simplify the machine, and possibly reduce damage to the crop. For hydraulic drive the two driving sprockets are mounted directly to the motor output shaft (i.e. a lay shaft is not necessary). [0119] It is desirable to design a base cutter and appropriate drives which will allow the shafts to run at an angle to the vertical. This angle should be adjustable over a range of several degrees and able to spring apart if an uncuttable obstruction enters between two rotating discs. In a preferred embodiment, the shape of the base cutter tips is adjusted to minimize the splitting of cane butts and resulting field losses. In addition, the blade should be detachable, with a shape that combines a sawing action with high resistance to stone damage. Vehicle tires, or a similar resilient structure, may be mounted onto the cutting discs in order to force the stalks out rearward. [0120] FIGS. 6 a and 6 b illustrate a general arrangement of pivots for two right-angle gearboxes, allowing the vertical shaft to be set at a predetermined angle to the direction of travel of the base cutter and/or allow the cutting discs to move apart. The drive arrangement 600 is a horizontal lay shaft 602 which drives two sprockets 611 (driving sprocket), 612 (driven sprocket), which in turn drive the horizontal shafts of each of two (handed) right-angle gear boxes 610. Reference 606 indicates the sense in which the vertical shaft 604 pivots. Each gearbox 610 is suspended in a frame constructed of approximately 5″ channel members 603 so that it can pivot around the axis of its horizontal shaft. The bearing housing of the gearbox horizontal shaft 616 pivots in a ring 614. Movable plates 602 which hold the gear pivots 616 are secured by four bolts 608. The pivoting structures can be moveable in the frame to allow different distances between the gearboxes and also for chain-tensioning purposes. [0121] FIG. 7 illustrates a restraining arrangement to set the angle of the vertical shaft. One pivoted member 702 may be attached to a bracket 700 welded to the gearbox 610, and another may be welded to a bracket 704 attached to the side frame 706. A threaded rod may be welded to the first pivoted member 702 and pass through a hole in the pivoted member in the side frame bracket 704. Suitable adjustable restraining devices, such as these brackets 700, 706, prevent the vertical shaft of each gearbox 610 from pivoting too close to the opposite unit, thus setting the distance apart of the two discs which carry the blades. [0122] Referring to FIG. 8, a spring loading device may force the vertical shafts together to resist normal opening forces in cutting the crop, but allows the cutting discs to move apart from uncuttable obstacles. Mounted with, or separate from, the restraining device is a device that may spring-load the two gearboxes, so that the vertical shafts are forced together. The strength of the spring-loading is chosen to prevent the cutting discs moving apart, due to the forces normally encountered in cutting the crop. But the setting is adjusted to allow the discs to move apart when a non-cuttable object is encountered. The spring-loading can be substituted by suitable hydraulic ram or rams, accumulator, and pump. In FIG. 8, a bridge structure 804 is welded on the pivoted member 702 and moves freely around pivoted member 708. The threaded rod 604 may be welded into the pivoted member 708. A pressure plate 800 tightens pivoted member 702 on the rod 604 to compress the spring 802. [0123] The relative degree of inclination of the two vertical shafts, the consequent spacing of the discs holding the cutting blades, and the force required to move the discs apart for an uncuttable object to pass through, may all be adjustable. Therefore the unit can be set to deal with a wide range of row shapes, crop conditions and field conditions. [0124] FIG. 9 shows a blade designed to combine the best features of replaceability with saw-type action. Mounted on the blade-supporting discs are a number of specially-designed, scimitar-shaped, notched blades 900 fabricated from high tensile steel. These blades are retained in sockets made from high tensile steel to resist wear if the blade chafes in the socket. A single retaining bolt prevents the blade from moving out of the socket by centrifugal force. A preferred embodiment may have 7 slots or notches equally spaced along the blade. [0125] Attached to the same blade-supporting discs may be one or more vehicle tires 1000 or tire-like structures ( FIG. 10 ) which touch, or nearly touch, in the center. These structures, meeting approximately in the center of the zone of horizontal cut, or cutting zone 1002, grip the cut stalks and push them rearwards. The tire structures may have a tilt of approximately 10 degrees 1004, consisting of inward and (a much smaller) forward tilt. The degree of resilience in the tire is controlled by either the choice of tire or by drilling holes of around 5-7 cm. diameter at intervals around the side walls of the tire. [0126] FIG. 11 shows the pivoted mounting of a gearbox 1100 onto a frame formed of two 5″ channel pieces 1102. The plates 1104 onto which the pivots 1106 are mounted, are held to the frame by four bolts 1108, allowing the distance between the driving sprocket and the driven sprocket 1110 to be adjusted. [0127] FIG. 12 shows the vertical axis of the gearbox inclined towards the centre of the machine and FIG. 13 shows it inclined outwards. Because the pivots 1106 are concentric to the bearing housings 1112 of the horizontal shafts of the gearboxes, the chain length remains constant throughout the angular movement of the vertical shaft. The driving 1200 and driven sprockets 1110 are shown. [0128] FIG. 14 shows the blade-holding discs 1402 mounted to the vertical shaft. A hexagonal structure 1404 surrounds the vertical shaft housing and rotates with it. This structure is referred to as the “drum”. At the upper end of the drum are three bearings positioned with their race casings touching a ring which forms part of the vertical shaft housing. In the embodiment shown in FIG. 15, the base cutter disc is connected to the drum by three resilient structures. These resilient structures are formed from steel plates 1502 with holes linked by rubber cylinder 1504 which is compressed by tightening a bolt 1506. Small ridges in the holes in the steel plates and matching grooves on the rubber cylinder 1504 (see also FIG. 16 ) ensure that the rubber 1504 does not rotate in the steel holes. [0129] FIG. 17 shows one possible restraining arrangement to set the maximum inward inclination of the vertical shaft. A pivoted member 1702 is attached to the gearbox. This pivoted member bears a threaded rod 1704 which passes through a hole in pivoted member 1706 on the side frame of the base cutter 1708. This side frame may itself be rigidly attached to the frame members holding the gearbox pivots 1702. By adjusting the position of nuts 1710, maximum inward inclination of the gearbox and its vertical shaft can be set. FIG. 18 shows an alternative restricting method with an adjustable bolt 1802, threaded into a plate 1804 which is welded to the gearbox and engages on the side frame. [0130] FIG. 19 shows one method of spring loading the two vertical shafts together 1902 in an inward sense. In this case it is integral with the restraining mechanism in FIG. 17. The pivoted member 702 mounted to the gearbox 610 bears a bridge structure 804 which holds a plate 1902 above the pivoted member 708 mounted on the side frame 706. A rod 604 attached to member 708 passes through the plate 1902 and one or more compression springs 802 are slipped over the rod 604 and tensioned downwards by a washer and nut turning on the threaded part at the top of the rod 604. [0131] FIG. 20 shows an alternative mechanism where adjustable springs 2002 act mutually on the two opposing gearboxes. Tension is increased by tightening nuts. A hydraulic mechanism may be used to achieve the same result. A ram pushes the top of the two gearboxes apart; an accumulator and hydraulic pump adjust the pressure exerted by the ram, which is forcing the tops of the gearboxes apart, and hence thrusting the vertical shafts together. [0132] The shape of the blades 2102 which attempt to combine the best of a sawing action and easy detachability, are best seen in FIG. 21 and in dimensioned drawings as shown in FIG. 9. These dimensions can be varied to suit available materials and details of the base cutter design. [0133] Mounting sockets for the blades are shown in FIG. 22. The socket is composed of high-tensile steel flats 2202 welded through holes 2204 onto the underside of the base cutter disc plates. A plate 2206 is welded across the flats and carries a hole for a retaining bolt 2208. The nut side of the bolt 2302, shown in FIG. 23, is guarded by sloping plates from being struck by crop stalks or stones, etc. [0134] FIGS. 26 and 27 show two views of the tires 2402 mounted to the base cutter discs; [0135] these tires increase the rearward movement of the cut stalks. FIG. 26 shows the walls of the tire drilled with holes 2602 to change its resilience. Feed Conveyor [0136] A feed conveyor with a pressure roll in the front on the top, may be used to convey stalks through the machine. FIG. 27 illustrates a conveyor 2702, brushes 2704 and feed roll 2706. The de-leafing of sweet sorghum stalks by brushing requires more exposure of the surface area than the de-leafing of sugar cane stalks by grabbing. In addition, sweet sorghum stalks require kinder treatment because they are less durable than sugar cane stalks. An additional feed roll may be added in the center of the conveyor 2702 to spread the stalks to give more area for the brushes to work and also reduces accumulated debris in the conveyor 2702 area. The conveyor 2702 may be resurfaced for better gripping the stalks. The conveyor can be extended to meet the lower brush to eliminate leaf loss, which also allows the extractor fan to be replaced with a simple blower. De-Trashing Roll [0137] A de-trashing roll, which may be located in the middle of the conveyor, is used to remove free trash, such as leaves, that are carried along the conveyor with the sorghum stalk. A feed roll may be added to the center of the conveyor by wrapping the conveyor with softer rubber sheets to meet the need for kinder conveying. Brush Rolls—Leaf Stripping Mechanism [0138] This section describes improvements to the harvesting of unburnt stalks of large grass crops like sugarcane and sweet sorghum. Means are described for removal of leaf material and its disposal, using brushes whose peripheral speeds considerably exceed the speed of the stalks which are moved through the machine by one or more restraining nips. Two brush rolls, one on top and another below, both at the end of the conveyor, sweep leaves from the stalks. The top roller center is forward and the rolls travel in the same direction as the conveyor but at a higher speed. Using this technique it is possible also (a) to use an extractor fan which projects the freed leaves forcibly onto the uncleaned stalks to increase the proportion of freed leaf material, and (b) that the conveying and cleaning of the crop can be effected with elements having their axes substantially vertical. [0139] There is interest in harvesting equipment which can cut and clean unburned sugarcane and crops like sweet sorghum without chopping the stalks into short pieces in order to remove the leaf material with extractor fans. [0140] The stripping of leaves in this machine is based on the acceleration of the stalk from a conveyor by a nip and the projection of that stalk between rubber fingers, over which a stream of air is directed. The air stream helps the ends of leaves to be engaged by a flail, which then forcibly pulls the leaves away from the stalk. [0141] An alternative method of cleaning stalks in a typical harvester, such as the well-known Centurion, is the subject of this application; the method could also be applied in an alternative machine also described herein. In particular, the disclosed invention restrains the movement of the stalks while they are being acted upon by brush elements moving appreciably faster than the stalk but in the same direction as its travel. The loosened material may simply fall away from the cleaned stalks but may also be removed by an extractor fan. Such fans are standard technology on “combine” cane harvesters, but in a preferred embodiment of the disclosed invention, the fan directs the removed material onto the stalks which have not yet passed through the final nip. With this mechanism the broken pieces of leaf become additional means of stripping material and cleaning the stalks, as well as depositing the material under the machine. [0142] The objectives of the leaf stripper are to use brush elements which are rotating to one or both sides of stalk material of crops like sugarcane or sweet sorghum to remove the leaves. Such brushes to rotate appreciably faster than the rate at which the stalk is being restrained by one or more conveying elements; to remove the loosened leaf material with an extractor fan, which forces the material back over the stalks before they are engaged by the final nip, and to exploit concept I in a vertically-oriented mechanism instead of the conventional horizontal orientation. [0143] FIG. 28 illustrates the general arrangement of a conveyor accepting cut stalks from a base cutting mechanism 2802, such as that described above. The conveyor 2804 has one or more restraining rollers 2806 on top of it which ensure that the crop passes up the conveyor at the same speed at which the conveyor belt is moving. Brushes are mounted on rotating elements 2808. Following the brush elements there may be a further pair of rollers 2810 forming a nip which grip the stalks and move them at a speed similar, though not necessarily identical, to the conveyor speed. An extractor fan 2812 may be installed to draw freed leaf material from around the stalks as they pass rearwards into a bin. The extractor fan is housed in a cowling 2814 which forces the air and free leaf material down onto the stalks before they enter the final nip. The broken leaf material bombards the stalks, further freeing leaf material from them. This arrangement also ensures that the unwanted leaf material is disposed of downwards—i.e. avoiding the nuisance of airborne material around the machine. [0144] Whole stalk cane harvesters, popularly called “soldier” or “Louisiana” carry the stalks through the machine in a more or less upright mode. On the other hand, cane harvesters popularly called “combine” harvesters, conventionally carry the stalks into the machine on a conveyor of which the driven shafts are parallel to the ground. Whole stalk machines for either cane or crops like sorghum, adopt the latter mode. However the leaf-removal mechanism described above can also be envisaged as being executed in a conveying system whose rotating axes are substantially vertical. [0145] For example, in FIG. 29 an arrangement is shown in which a base cutter 2902 feeds a first nip 2904 formed of a number of vehicle tires on two vertically-rotating, opposed shafts. Brushes 2906 act on the stalks at a peripheral speed appreciably greater than the nip speed. A second nip 2908 rotates with a peripheral speed similar, though not necessarily identical, to the first nip 2904 and conveys the stalks to a receptacle 2910 for periodic discharge. Extractor fans may be suitably placed (e.g. to each side of, and slightly rearward of, nip 2908 if desired. [0146] FIG. 30 shows an approximate scale drawing of a conveyor 2804 and two rollers 2806 which press the crop onto the conveyor, ensuring that it goes at the same speed as the conveyor belt is moving and cannot be easily accelerated whilst the brushes are acting upon it. [0147] The brush elements 2906 as shown in FIG. 31 may be formed from plastic cleaning bristles commonly used to clean conveyors. The brush may be driven hydraulically and its speed may be adjustable, but is typically 3-4 times the peripheral speed of the conveyor and nip 2904. FIG. 32 shows nip 2810 formed from rubber-covered rollers which accept the stalks after they pass through the brushes and project them into a receptacle. [0148] FIG. 33 shows the extractor fan 2812 and cowling 2814 mounted as in FIGS. 28 and 30. [0149] The fan may be hydraulically driven and may be typical of extractor fans used on sugarcane “combine” harvesters. [0150] FIG. 34 shows an alternative arrangement in which there are no nip rollers after the brushes; the brush elements 2808 are envisaged in this case as being of adequate power to project the crop into the receptacle, as well as cleaning off the leaf material. [0151] The novel leaf stripping mechanism uses brushes to sweep leaves off sweet sorghum stalks. The harvester's cleaning device involves the most development as it is being optimized for clean removal of leaves and other trash while minimizing stalk damage. The cleaning brushes are constantly optimized for removal and longer life. Hurler Drums [0152] A preferred embodiment of a harvester modified with hurler drums typically is fitted with two hurler drums; two rolls (an upper and a lower) of equal diameter located perpendicular to the conveyor belt. Hurler drums finish pulling cane through brush rollers and “hurl” the stalk into the bin and head topper. Proper hurling of the thin sweet sorghum stalks requires hurler drums made of soft sheets of rubber. Guiding plates are configured to the full length of the hurler and for the relative geometry of the hurler, conveyor, and brushes. The rear axle must leave adequate exit for trash due to hurler movement. [0153] The rear axle supports are configured to open up an area to allow access to hurler rollers and brush rollers. This configuration allows for more open space for debris to be directed to the ground and to provide access for maintenance. In using this configuration, it was prudent to reinforce it in another area to keep structural integrity intact. This modification prevents the build up of debris that may impede clearing the debris from the processing of the crop. [0154] The lower hurler roller, shown in FIG. 35 is configured to allow mounting of the treaded bonded-rubber panels to increase grip on stalks. The top hurler roller provides softer pressure to compensate for the harder lower hurler FIG. 36. Softer tires/rubber panels 3602 are installed on the upper hurler. FIG. 37 illustrates the soft rubber wheels 3702 that will cover the upper hurler roller. The center tires engage special plates in center of the driver structure that are used to space that are used to add some rigidity to the structure and prevent drifting of the tires. The lower hurler in FIG. 38 uses hard tires/rubber 3802 similar to those used on sugar cane harvesters. [0155] FIG. 39 shows the reworked chain case 3902 to cover the new drive and give quicker access to hurler drives. FIG. 40 shows repaired top hurler pivots 4002 and devised lubrication system to prevent a wear problem. De-bridging Flail Roller [0156] A de-bridging flail roller with comb fingers and an air blast catches and ejects any remaining trash downwards after the stalk heads are cut. The de-bridging roller may be located just behind the head topper. One construction of a de-bridging thrash roller is a shaft with arms with chain link attached. The roller must be modified for kinder handling and to compensate for modifications made to the air blast. FIG. 41 shows a de-bridging flail 4102, stabilizing clamps 4104 and side breakers 4106. Air Blast [0157] The air blast, from a fan pulling air near the engine, is directed downwards towards the flail to separate trash from stalks, blowing leaves to the ground. For sweet sorghum harvesting, the air blast must be modified to account for the smaller and lighter leaves of sweet sorghum compared to sugar cane. Shown in FIG. 42, a deflector 4202 pushes leaves into the front part of the bin. The fan may be hydraulically driven 4302, as shown in FIG. 43, with an intake at the brush rollers and output between the hurlers and a de-bridging thrash roller. A chain case covers the drive and gives quick access to the hurler drives. A modified cover for the top brush 4402 shown in FIG. 44 allows leaves to be sucked off by fan. FIG. 45 shows the reworked entry (previously exit) 4502 of extractor fan. FIG. 46 shows the rubber belting 4602 cover linking the top brush area to the fan entry. The cover folds back or easily removes to allow access to engine. FIG. 47 shows the reworked fan outlet 4702. FIG. 48 shows the belting 4802 attached to the fan, prior to installation. [0158] The fan airflow has been reversed so air is blown instead of sucked. The fan has reverse rotation of the blower. The fan assembly is moved back to pull air from just past brush rollers, instead of near the engine, and after deflection blows down in front of the debridging flail. The fan tube structure on top of the machine runs from in back of the engine to the full length of the roller brushes (like a half fender on a bicycle). The inlet pulls above brush roller and expels behind rear hurlers. The fan discharges between the hurlers and the cutter head above the debridging flail. A sheet of plywood deflects air flow to the back side. Rubber belting covers 4802 link the top brush area to the fan entry. The cover folds back or easily removes to allow access to the engine. The air flow is controlled with rubber sheeting to keep air sealed on side above the engine. The loop was closed for suction and the sheeting flipped up for access to the engine during maintenance. The air was pulled through and blown out on top hurlers. [0159] FIGS. 49 and 50 illustrate the modified axle support in front 4902 and rear 5002 view along with the cover behind the axle, and detail of the corners and cut-off bar. As illustrated in FIG. 51, this structure 5102 may be located below the lower hurler. Toppers [0160] Two toppers, comprising of a pair of sharp-toothed discs rotating outwards against ledger plates, sever the tops of the stalk to length and remove the valuable grain heads from the stalk. Toppers are made adjustable fore and aft to accommodate varying stalk heights and desired cutting length of head and stalk. The topper must be modified to account for the smaller and much different tops (grain heads) compared to sugar cane tops. FIG. 52 shows the topper unit 5202 adjusted back into the bin and re-hosed from below to permit free movement. The fan was moved to make room for the topper. A rubber sheet may be mounted in front to form separate piles of crop components. The topper structure 5302 is shown in FIG. 53. To accommodate the bin extension, the topper may be moved back. A reason for this change is that cane tops are leaves, considered trash that are blown onto the field while sweet sorghum tops are valuable grain seeds. Bin [0161] The bin is the storage area at the rear of the machine; it accumulates and discharges the stalks and leaves into piles. It may be split to collect two separate piles of stalks, used for ethanol processing, at the back of the bin and leaves and heads, used for silage animal feed, at the front of the bin. Alternatively, only the stalks, used for ethanol processing, and heads, used for grain feed, could be collected in the two separate sections of the bin with the leaves blown onto the field. Or the bin could be separated into three sections with the leaves collected separately from the heads, for use as fuel for the process offseason, while the stalks are used for ethanol processing and the heads are used for grain feed. The bin of a sugar cane harvester must be modified to account for longer and thinner stalks of sweet sorghum compared to sugar cane. FIG. 54 shows the bin frame extended with a bin extension 5402 to allow for collection of 12 to 13 foot stalks. FIG. 55 shows the telescope section of the bin 5502 completely remodeled to allow 12 foot stalks and increase capacity. FIG. 56 shows new sidings 5602 for the front of the bin. FIG. 57 shows the axle cover 5702, sidings to the topper track 5704, bin door extensions 5706 and mid-bin rubber sheet and support 5708. FIG. 58 shows the rubber sheet 5802 that separates the front and rear parts of bin. The bin is typically equipped with operator controlled doors for dropping harvested sorghum into piles on the ground. A cushion 5902, shown in FIG. 59, may be used to replace rollers supporting the bin doors to stop banging of the door when the row direction is changed. The bin may adjust in length to the height of the stalk, to cut the seed head from the stalk to length as the height of the crop changes. A cable is extended from the back of the bin to the cab window so the driver can use the height of the stalks to set the length of the bin and toppers. When the butts of the stalks hit the back of the bin and fall on the topper, the tops are separated into the front part of the bin and the stalks to the back. [0162] FIG. 60 depicts the catch rubber ( FIG. 60 a 6002 ) and the weight ( FIG. 60 b 6004 ) that keeps the catch-rubber forward in for the first few stalks. FIG. 61 shows extensions to the doors 6102 for the special front compartment. FIG. 62 shows the main axel beam area 6202, which was reworked (again) to improve access to the conveyor, hurler, and brushes, including by mounting a big cross-tube. [0163] It is to be understood that even though numerous characteristics and advantages of various embodiments of the present invention have been set forth in the foregoing description, together with details of the structure and function of various embodiments of the invention, this disclosure is illustrative only, and changes may be made in detail, especially in matters of structure and arrangement of parts within the principles of the present invention to the full extent indicated by the broad general meaning of the terms in which the appended claims are expressed. For example, the particular elements may vary depending on the particular application of the harvesting system that are organized or designed differently while maintaining substantially the same functionality without departing from the scope and spirit of the present invention. | Summary: The disclosed invention relates to the harvesting of sugar-bearing ground crops such as sweet sorghum, in cold regions. The sugar crop harvester with various adaptations described is comprised of operational components, or features, that minimize the damage done to the crop during harvesting, particularly those things that result in deterioration of sugars prior to processing. The harvester includes several operational components that are well suited for processing sweet sorghum, particularly the dividers, base cutters, feed conveyor, de-trashing roll, brush rolls, hurler drums, de-bridging thrash roller, air blast, toppers and bin. Other components of the harvester are designed for easy maintenance. The operational components can be used with either a whole-stalk style harvester, or a chopper-style harvester. | 13,689 | 176 | big_patent | en |
Summarize: Procedural Adjustments The Reauthorization Act changes procedures associated with federal capital cases including those relating to air piracy cases arising before 1994 and habeas procedures for state capital petitioners. Pre-1994 Capital Air Piracy Cases In the early 1970s, the U.S. Supreme Court held unconstitutional the imposition of capital punishment under the procedures then employed by the federal government and most of the states. In 1974, Congress established a revised procedure for imposition of the death penalty in certain air piracy cases. In 1994, when Congress made the procedural adjustments necessary to revive the death penalty as a sentencing option for other federal capital offenses, it replaced the air piracy procedures with those of the new regime. At least one court, however, held that the new procedures could not be applied retroactively to air piracy cases occurring after the 1974 fix but before the 1994 legislation, in the absence of an explicit statutory provision. The Reauthorization Act adds an explicit provision to the end of the 1994 legislation. H.R. 1763 and H.R. 3060 contain comparable provisions. Habeas Corpus in State Capital Cases Federal law provides expedited habeas corpus procedures for state death row inmates in those states that qualify for application of the procedures and have opted to take advantage of them. As of enactment of the Reauthorization Act apparently, few if any states had sought and been found qualified to opt in. Under the Reauthorization Act, states opt-in or have opted-in as of the date, past or present, upon which the Attorney General determines they established or have established qualifying assistance of counsel mechanism. The earlier provision required that the mechanism include competency standards for appointed counsel. The Reauthorization Act removed the requirement, but granted the Attorney General regulatory authority sufficient to establish such standards. The act establishes a de novo standard of review for the Attorney General's determination before the D.C. Circuit. The Streamlined Procedures Acts in the House and Senate, H.R. 3035 and S. 1088, would make similar changes in the opt in procedure. S. 956, H.R. 2388, and H.R. 3132 (House-passed legislation is noted in italics), contain a common amendment governing federal habeas cases of an individual convicted under state law of killing a child, proposed 28 U.S.C. 2254. Habeas under section 2254 would have been unavailable in such cases except for claims that both (1) relied on a new constitutional interpretation made retroactively applicable by the Supreme Court or on evidence that the petitioner could not reasonable have been previously discovered and (2) were predicated upon facts in the face of which no reasonable judge or jury would have found the petitioner guilty but for the constitutional error, proposed 28 U.S.C. 2254(j)(1), (2). Under the bills, judicial consideration of claims that meet the dual criterion would have been expedited. Additional Procedural Proposals: Venue Several anti-gang bills purport to change the place where capital cases may be tried. S. 155, H.R. 970, H.R. 1279, and H.R. 4472 contain the same provision that would have rewritten 18 U.S.C. 3235. Section 3235 provides that where possible capital cases should be tried in the county in which the crime occurred. The proposal would have repealed the "county trial" language of section 3235 and replaced it with language reminiscent of the multi-district terms of section 3237(a): "(a) the trial of any offense punishable by death shall be held in the district where the offense was committed or in any district in which the offense began, continued, or was completed. (b) If the offense, or related conduct, under subsection (a) involves activities which affect interstate or foreign commerce, or the importation of an object or person into the United States, such offense may be prosecuted in any district in which those activities occurred," proposed 18 U.S.C. 3235. The proposal would have operated subject to two constitutional provisions and two Supreme Court cases which construe them. Mitigating and Aggravating Factors The death penalty may be imposed in a federal capital case only after consideration of the mitigating and aggravating factors listed in 18 U.S.C. 3592 and only if at least one aggravating factor is found. Several bills would have adjusted the factors. One of the aggravating factors in homicide cases consists of the fact that the death resulted from the commission of a list of designated felonies. The Adam Walsh Child Protection and Safety Act adds 18 U.S.C. 2245 to the list, 18 U.S.C. 3591(c)(1). Among the proposals that failed to secure final passage, H.R. 3860 would have added 18 U.S.C. 2245 (sexual abuse resulting in death) to the list, proposed 18 U.S.C. 3591(c)(1). H.R. 3060 would have placed 18 U.S.C. 2339D (receipt of military training from a foreign terrorist organization) on the list, and H.R. 5040 would have done the same and also added 18 U.S.C. 241 (civil rights conspiracy), 245 (deprivation of federally protected activities), 247 (interference with religious exercise), 1512 (tampering with federal witnesses), and 1513 (retaliating against federal witnesses), proposed 18 U.S.C. 3592(c)(1). Both H.R. 3060 and H.R. 5040 would have made obstruction of justice an aggravating factor in homicide cases, proposed 18 U.S.C. 3592(c)(17). Other Procedural Proposals H.R. 3060 would have allowed the court upon a finding of good cause or agreement of the parties to proceed with a capital sentencing jury of fewer than 12 members, proposed 18 U.S.C. 3593(b). Existing law requires agreement of the parties. The bill also amends Rule 24(c) of the Federal Rules of Criminal Procedure to allow for the selection of a maximum of 9 alternate jurors and allows each side 4 peremptory alternate juror challenges when either 7, 8, or 9 alternates are to be selected, proposed F.R.Crim.P. 24(c). The present Rule calls for a maximum of 6 alternates and affords the parties 3 alternate juror peremptory challenges. These and other similar proposals passed the House initially as part of H.R. 3199, but were dropped in conference and were not part of the Reauthorization Act as passed. H.R. 5040 would have struck the provision which outlaws the execution of the mentally retarded, proposed 18 U.S.C. 3596(c). The omission, although perhaps surprising to some, appeared inconsequential since execution of the mentally retarded is constitutionally proscribed. The bill also would have required notice to the government and would have permitted the government to request an independent mental health examination when a defendant intends to enter mental retardation as a mitigating factor for capital sentencing purposes, proposed 18 U.S.C. 3593(b). The existing statute mentions no such requirements. Presumably recourse to the proposed procedure would have been more infrequent in those cases where the district court conducted a pre-trial evidentiary hearing to determine whether the mental retardation of the accused precluded imposition of the death penalty following any conviction. Present law permits a capital jury to unanimously recommend a sentence of death or of imprisonment without possibility of release; if they do not, the court is to sentence the defendant to any lesser sentence authorized by law, i.e., imprisonment for life or a term of years. H.R. 5040 would have provided that if the jury cannot agree on a capital recommendation, a new sentencing jury would have been empanelled and the issue retried, proposed 18 U.S.C. 3594. Existing law specifically contemplates that the execution of federal capital sentences will be carried out in state facilities. H.R. 5040 would have granted the Attorney General regulatory implementing authority without exclusive reference to state facilities, proposed 18 U.S.C. 3596, 3597. The bill also would have rewritten 18 U.S.C. 3005 which assures defendants two assigned counsel in capital cases. The proposal would have made it clear that the statute only applied when the government sought the death penalty and not in capital cases where it had elected not to do so, proposed 18 U.S.C. 3005(a). The federal appellate courts are divided on the question over whether section 3005 now entitles a defendant to the assistance of two attorneys in all capital cases or only in those in which the government actively seeks the death penalty. The proposal also would have explicitly authorized the government to strike for cause potential jurors in capital cases whose opposition to the death penalty "would prevent or substantially impair the performance" of their duties as jurors, proposed 18 U.S.C. 3005(b). The proposal borrowed language from Supreme Court cases indicating that a potential juror may be struck if his views on capital punishment "would prevent or substantially impair the performance of his duties as a juror in accordance with his instructions and his oath." New Federal Capital Offenses Title III of the Reauthorization Act, designated the Reducing Crime and Terrorism at America's Seaports Act, creates three new federal capital offenses: (1) 18 U.S.C. 2282A (devices or dangerous substances in waters of the United States likely to destroy or damage ships or to interfere with maritime; causing a death); (2) 18 U.S.C. 2283 (transportation of explosive, biological, chemical, or radioactive or nuclear materials; causing a death); and 18 U.S.C. 2291 (destruction of vessel or maritime facility; intentionally causing a death). Two other port security bills would have suggested similar new death penalty offenses, H.R. 2651 and S. 378 (as reported) (proposed 18 U.S.C. 2282A, 2283, and 2291), and a third would have offered three slightly less comparable offenses, H.R. 173 (proposed 18 U.S.C. 1372 (destruction of vessel or maritime facility; if death results), 2280A (devices or substances in waters of the United States likely to destroy or damage ships; if death results), and 2282 (malicious dumping; if death results)). The bills drafted to counter gang violenceâ H.R. 4472, H.R. 1279, H.R. 970, and S. 155 âfrequently include two new federal death penalty offenses. One of the proposed offenses would have proscribed the use of interstate facilities with the intent to commit multiple murders and would have been a capital offense where death results. The other, modeled after the provision that condemns the use of a firearm during or in relation to a crime of violence or a drug offense, would have outlawed crimes of violence committed during or in relation to a drug trafficking offense and makes the offense punishable by death if a death results. A few other bills would have made it a federal capital offense to kill a police officer under various circumstances. For example, H.R. 2363 would have outlawed killing a peace officer and fleeing the country, proposed 18 U.S.C. 1121(c). H.R. 1751 and H.R. 2194 would have prohibited murdering federally funded state or local law enforcement officers, proposed 18 U.S.C. 1123. Other proposed new federal capital offenses would have included (1) agroterrorism when death results, proposed 18 U.S.C. 2339D ( S. 1532 ); (2) interference with federal disaster relief efforts if death results, proposed 18 U.S.C. 1370 ( H.R. 3728 ); (3) death resulting from a violation of 18 U.S.C. 1590 (trafficking in persons) that involves raping or kidnapping more than one person, proposed 18 U.S.C. 1590 ( S. 2437 ); (4) death resulting from a violation of proposed 18 U.S.C. 555 ( S. 2611 ) that would have proscribed evading immigration, customs or agricultural inspection at the border; and (5) death resulting from the commission of federal crimes of terrorism, violations of 18 U.S.C. 175 (biological weapons), 175b (biological materials), 229 (chemical weapons), 831 (nuclear materials), or of 42 U.S.C. 2284 (atomic weapons), or conspiracies or attempts to commit such crimes or violations, proposed 18 U.S.C. 2339E ( H.R. 3060, H.R. 5939, S. 3882, S. 3848 ). Capital Punishment for Violation of Existing Crimes Section 110 of the Reauthorization Act merges 18 U.S.C. 1992 (2000 ed.) (wrecking trains) and 18 U.S.C. 1993 (2000 ed.) (attacks on mass transit) into a new 18 U.S.C. 1992. The train wreck offense was a capital offense; the mass transit offense was not; under the new section both are now capital offenses, 18 U.S.C. 1992. The most common example of a proposed death penalty sentencing option for an existing crime comes from some of the child safety bills, many of which would have made the death penalty available where a child dies as a result the commission of a federal crime of violence or some other federal crime: (1) S. 956 (crime of violence, proposed 18 U.S.C. 3559(d)); (2) H.R. 2388 (same); (3) H.R. 3132 (same); (4) H.R. 4472 (same); and (5) H.R. 3860 (violations of 18 U.S.C. ch.110 (sexual exploitation of children), ch. 117 transportation of illegal sexual activity), or 1591 (sex trafficking in children), proposed 18 U.S.C. 2245(b)). Congress adopted a variation of this theme in the Adam Walsh Child Protection and Safety Act when it amended 18 U.S.C. 2245 to make murder a federal capital offense when committed in the course of a wider range of federal child sexual abuse offenses. The gang bills generally would have rewritten the federal criminal gang statute (18 U.S.C. 521) to permit imposition of capital punishment for a death-resulting violation of the newly crafted provisions or of the Travel Act (18 U.S.C. 1952): H.R. 1279, proposed 18 U.S.C. 521, 1952; S. 155, proposed 18 U.S.C. 523, 1952; H.R. 4472, proposed 18 U.S.C. 521, 1952; see also, H.R. 970, proposed 18 U.S.C. 523. H.R. 3060, H.R. 5939, S. 2883, and S. 3848 would have made capital offenses of several death-resulting terrorism-related offenses that are now punishable by no more than life imprisonment, specifically, proposed 18 U.S.C. 832 (participating in foreign nuclear or other weapon of mass destruction programs), proposed 18 U.S.C. 2332g (anti-aircraft missiles), proposed 18 U.S.C. 2332h (radiological dispersal devices), proposed 18 U.S.C. 175c (smallpox virus), and proposed 18 U.S.C. 42 U.S.C. 2272 (atomic weapons). It is possible that the drafters of H.R. 3060 also intended to treat receipt of military training from a foreign terrorist organization, 18 U.S.C. 2339D, like treason and espionage; that is, to make it a capital offense even if no death results from commission of the offense. The statutes that outlaw treason and espionage make them punishable by death or a term of imprisonment, 18 U.S.C. 2381, 794. Section 3591(a)(1) of the federal capital punishment procedures provides that treason or espionage are punishable by death if execution is found justified after considering the mitigating and aggravating factors listed in section 3592. Section 3592(b) lists three aggravating factors for treason and espionage cases, i.e., (1) the offender has a prior espionage or treason conviction, (2) the offense involved a grave risk to national security, and (3) the offense involved a grave risk of death. Violation of section 2339D is punishable by imprisonment for not more than 10 years, 18 U.S.C. 2339D(a). H.R. 3060 would have made no change in section 2339D, but it would have amended section 3591(a)(1) of the capital procedures provisions to say that violations of sections 2381 (treason), 794 (espionage), or 2339D (terrorist training) may be punished by death if execution is found justified after considering the mitigating and aggravating factors listed in section 3592, proposed 18 U.S.C. 3591(a)(1). It also would have amended the list of 3592(c) aggravating factors to add a fourth factor, i.e., the defense involved substantial planning by the defendant, proposed 18 U.S.C. 3592(c)(4). Assuming the conforming amendment to section 2339Dâmaking it a capital offenseâwas an oversight and in spite of the proposal's caption ("addition of terrorism to death penalty offenses not resulting death"), it is not clear that the courts would permit imposition of the death penalty for a violation of section 2339D unless the offense also involved a first degree murder. The Eighth Amendment's cruel and unusual punishment clause precludes imposing the death penalty for the rape of an adult woman by an individual already under a sentence of life imprisonment at the time of the rape; it precludes imposition of the death penalty even in the case of murder unless the defendant at least acted intentionally or acted with reckless indifference to human life while participating in a felony involving a murder; and since the Court's decision in Furman v. Georgia, it has never been called upon to approve, and consequently has never approved, imposition of the death penalty for a crime that did not involve murder. Moratorium H.R. 4923 / S. 122 would have repealed federal death penalty provisions and barred imposition or execution of any capital sentence for violation of federal law. It made no mention of capital punishment imposed for violation of state law. H.R. 379, on the other hand, would have set a ten year moratorium on imposition and execution of capital sentences in any state in which an individual originally sentenced to death had subsequently been judicially found innocent. It said nothing of capital punishment imposed or executed under federal law. | Summary: The USA PATRIOT Improvement and Reauthorization Act (Reauthorization Act), P.L. 109-177, 120 Stat. 192 (2006) contains a number of death penalty related provisions. Some create new federal capital offenses making certain death-resulting maritime offenses punishable by death. Some add the death penalty as a sentencing option in the case of pre-existing federal crimes such those outlawing attacks on mass transit. Some make procedural alterations such as those governing federal habeas corpus provisions for state death row petitioners. Other proposals offered during the 109 th Congress followed the same pattern: some new crimes; some new penalties for old crimes; and some procedural adjustments. Other than the Adam Walsh Child Protection and Safety Act, P.L. 109-248, 120 Stat. 587 (2006), none of the other proposals were enacted, although one House or the other approved several. Among these, H.R. 1279 would have amended the venue provision for capital cases and made it a federal capital offense to use the facilities of interstate commerce to commit multiple murders and another to commit murder during and in relation to a drug trafficking offense. As would have H.R. 4472. H.R. 1751 and H.R. 4472 would have made it a federal capital offense to murder a federally funded public safety officer. H.R. 3132 would have created special expedited habeas review of state child murder cases. And S. 2611 would have made murder committed during the course of certain federal offenses a capital offense. Of the capital proposals pending at adjournment, H.R. 4923 and S. 122 would have abolished the death penalty as a federal sentencing alternative and H.R. 379 would have imposed a moratorium barring the states from imposing or carrying out the death penalty. This is an abridged version of CRS Report RL33395, The Death Penalty: Capital Punishment Legislation in the 109 th Congress, by [author name scrubbed] (pdf), without the footnotes, appendices, or most of the citations to authority found in the longer report. | 4,941 | 528 | gov_report | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This patent application claims priority from, and expressly incorporates by reference, the following provisional patent applications: 60/659,226—Shielding Apparatus for Locking onto a Needle—filed on Mar. 7, 2005; 60/659,217—Needle Shielding Apparatus with Tubular Needle Cover—filed on Mar. 7, 2005; 60/659,213—Needle Shielding Apparatus with Tether to Needle Hub—filed on Mar. 7, 2005; 60/714,954—Blood Collection Device with Needle Shield—filed on Sep. 7, 2005. BACKGROUND [0006] This patent application describes and relates to medical devices for collecting blood or other bodily fluids or infusing fluids, such devices using needles to pierce a human or animal body. It includes a device for shielding such needles. SUMMARY OF THE INVENTION [0007] An embodiment of the invention is a winged needle set which has a housing with at least one outwardly extending wing (preferably two). A finned member is secured to the housing. The finned member is oriented generally radially outward relative to the longitudinal axis of the needle and is rotatable relative to the housing from a first non-shielding position to a second shielding position. A needle shield assembly is mounted relative to the housing such that the needle shield assembly is slidable longitudinally along the needle. When the finned member is rotated from the first non-shielding position to the second shielding position, the needle shield assembly is unlocked by a release mechanism and permitted to slide relative to the housing along the needle shaft, shielding the needle. In the first non-shielding position, the finned member is oriented from approximately vertical to approximately 60 degrees from vertical. The wing is moveable and can be brought towards the finned member (or rotated or folded) when the finned member is in the first non-shielding position. A spring biases the needle shield assembly to the second shielding position. The wings may be rotatable relative to the housing such that the wings can be brought towards the fined member when the finned member is in the first non-shielding position. BRIEF DESCRIPTION OF THE DRAWINGS [0008] FIG. 1 is an isometric view of a device incorporating the invention before deployment of the needle shield; [0009] FIG. 2 is an isometric view of a device incorporating the invention after deployment of the needle shield; [0010] FIG. 3 is a cross-sectional side view of a device incorporating the invention before deployment of the needle shield; [0011] FIG. 4 is a cross-sectional isometric view of a device incorporating the invention before deployment of the needle shield; [0012] FIG. 5 is a cross-sectional side view of a device incorporating the invention after deployment of the needle shield; [0013] FIG. 6 is a cross-sectional isometric view of a device incorporating the invention after deployment of the needle shield; [0014] FIG. 7 is a side view of a device incorporating the invention before deployment of the needle shield; [0015] FIG. 8 is a cross sectional view through section A-A in FIG. 7. [0016] FIG. 9 is an isometric view of part of the needle shield actuator of a device incorporating the invention; [0017] FIG. 10 is an isometric view of the distal end of part of a needle shield used in a device incorporating the invention. DETAILED DESCRIPTION [0018] The following is a description of the preferred embodiment of the invention as applied to a blood collection device. A similar structure may be used for infusing fluids. The purpose of the blood collection device 5 is to pierce a blood vessel (or other organ) using needle 10 and remove blood (or other fluid) to a receptacle via tube 15. This device makes use of technology described in U.S. Provisional Patent Application No's 60/659,213, 60/659,217 and 60/659,226 which are incorporated herein by reference. [0019] The preferred embodiment of the device is made up of the following basic components: [0020] Housing 20, with wings 225 and 230. [0021] Needle 10, secured to needle hub 50 and in fluid communication with tube 15. [0022] Needle shield assembly 110. [0023] Actuator assembly 30, with rotatable fin 300. [0024] Housing 20 has a generally cylindrical body 200, having distal end 205 and proximal end 210. Distal end 205 has an opening 215. Proximal end 210 has an opening 220. A passageway 236 extends between the openings in the proximal and distal ends. Passageway 236 is dimensioned such that shield assembly 110 fits axially in it, and such that shield assembly 110 can slide axially along it. Housing 20 is provided with wings 225 and 230, which can bend upwards towards each other and towards fin 300 on actuator assembly 30. Fillet 227 facilitates molding of wings 225 and 230 and housing 20. An identical fillet is provided to stabilize wing 230. Wings 225 and 230 are provided with indentations 250 and 255 respectively. These are shaped and dimensioned to accommodate fin 300. Housing body 200 is provided with a slot 260, designed to accommodated key 190 on shield assembly 110 (described below). Slot 260 extends from proximal end 210 (where it is open) towards distal end 205 of housing body 200, where it is closed. Slot 260 has a proximal end 265 and a distal end 270. [0025] Needle hub 50 has a stepped distal end 550 forming a hollow open ended cylinder 555 which mates with opening 215 in proximal end 210 of housing body 200. The step forms a flange 570. Stepped proximal end 550 is provided with a slot 560 (see FIG. 6 ), which aligns with slot 260 and extends from flange 570, along cylinder 555 to its open end. Slot 560 also accommodates key 190. Slot 560 is open at the front to allow key 190 to move in it and closed at the back to prevent key 190 from leaving it. [0026] Proximal end 75 of needle 10 is glued into needle hub 50. Rear end 565 is an open ended cylinder extending proximally which is designed to mate with tube 15, thus permitting fluid to flow through needle 10 and tube 15 and into a receptacle for collection. [0027] Fin assembly 30 has a cylindrical body section 350 having a proximal end 365 and a distal end 360. Proximal and distal ends 365, 360 are respectively provided with openings 366 and 367. A passage 370 extends between the proximal and distal ends. Passage 370 has an inner surface 372. Fin assembly 30 fits concentrically over cylindrical body 200 and can rotate about the axis of cylindrical body 200, constrained by wings 225 and 230. Near proximal end 365, inner surface 372 is provided with a circumferential rim 375. Rim 375 has sections removed from it forming openings 380 and 382 (only 380 is shown, 382 being substantially identical to 380 ), dimensioned to allow passage of key 190. These openings start at about + 70 degrees and −70 degrees to the vertical and describe arcs of about 30 degrees. When fin assembly 30 is in a first position, rim 375 blocks key 190. In that first position, when key 190 is blocked by rim 375, fin 300 is vertical. When fin 300 is rotated clockwise or counter-clockwise, cylindrical body section 350 (and hence rim 375 ) rotates about the axis of body section 200. When fin assembly 30 is rotated to a second position (anywhere from about 60 degrees to the vertical to about 90 degrees to the vertical, clockwise or counter-clockwise) in which key 190 aligns with opening 380 or 382 (depending on the direction of rotation) key 190 is free to travel distally in slot 260. When that happens, under the influence of coil spring 180 abutting inner wall 114 of cylindrical body 112, needle shield assembly 110 will move in the distal direction, thus shielding the needle. [0028] The combination of rim 375 and key 190 thus forms part of a triggering mechanism which allows shield assembly to be unlocked and to move in the distal direction when needle 10 is to be shielded. [0029] When fin 300 has been rotated to a position against either wing 225 or wing 230, fin 300 may be secured to the skin of the patient. To that end, fin 300 may be provided with an adhesive strip. Needle shield assembly 110 has a cylindrical body 112 having a proximal end 120 and distal end 115. Lumen 117 extends between proximal end 120 and distal end 115 and is dimensioned to accommodate needle 10 axially such that it can slide over needle 10. Needle shield assembly 110 fits axially into housing body 200 such that it can slide axially along passageway 236. Proximal end 120 is provided with key 190. As described above, key 190 fits into slot 260 and slot 560. Key 190 prevents shield assembly 110 from rotating relative to housing body 200. It also prevents shield assembly 110 from exiting opening 220 of housing body 200 in the proximal direction, when it abuts distal end 270 of slot 260. [0030] At distal end 115 of needle shield assembly there is a stop mechanism 116 for preventing shield assembly from sliding backwards once needle 70 is shielded. Proximal end 115 has a stepped section 150, i.e. a region of reduced diameter in comparison with the remaining cylindrical part of cylindrical body 112. Lumen 117 also has a reduced diameter in stepped section 150 compared with the diameter of lumen 117. In this region, lumen 117 is referred to as lumen 118. The diameter of lumen 11 8 is only slightly larger than the outer diameter of needle 10. Coil spring 111 is threaded over stepped section 150. The rear end of coil spring 111 abuts the wall 114 formed at the intersection of stepped section 150 and the remainder of cylindrical body 112. Coil spring 111 is a compression spring which exerts its force axially in the proximal and distal directions. Stepped section 150 is also provided with opening 160 in the form of a specially shaped slot extending in a distal direction approximately from the mid point of stepped section 150 to distal end 115 of cylindrical body 112 (see FIG. 10 ). This opening 160 is dimensioned and shaped such that ball 122 rests in it, with part of ball 122 extending into lumen 118 of cylindrical body 112 and abutting outer surface 11 of needle 10. The force in coil spring 111 keeps ball 122 pressed against needle 10. Shield assembly 110 can thus slide along needle 10 with very low frictional force. Opening 160 is also dimensioned and shaped such that when ball 122 no longer abuts needle 10 (i.e. the tip has passed ball 122 ), ball 122 can move distally towards distal end of 115 of cylindrical body 112 and radially further into lumen 118, thus blocking axial movement of needle 10 in the distal direction. This is described in greater detail below. [0031] Cap 100 is a metal stamping, dimensioned to fit over stepped section 150, thus enclosing coil spring 111. Cap 100 is provided with opening 170, dimensioned such that part of ball 122 can fit into it, when needle 10 abuts ball 122, but such that ball 122 cannot escape through it. Cap 100 may be limited in size to fit over distal end 115 of cylindrical body 110 or it can extend along the entire length of cylindrical body 115 as a unitary sheath. [0032] Behind stepped section 150, within lumen 117 of cylindrical body 112 lies coil spring 180. Coil spring 180 is a compression spring whose force is exerted axially in the proximal and distal directions. Distal end 191 of coil spring 180 abuts the back of inner wall 114 just behind stepped section 150. Proximal end 195 of coil spring 180 abuts proximal face 552 of needle hub 50 (i.e. in the region of flange 570, but inside cylinder 555. Coil spring 180 is therefore trapped within cylindrical body 112 of shield assembly 110 and inside housing 20. When shield assembly 110 is in its un-actuated position, coil spring 180 is compressed. [0033] The operation of stop mechanism 116 will now be described. Needle tip 70 has a beveled tip with two bevels, first bevel 71 and second bevel 72. When needle shield assembly 110 slides along the length of needle 10 in the distal direction, ball 122 aligns with bevel 71. When ball 122 encounters bevel 71, it is less radially constrained by needle 10 and it moves radially towards the axis of needle 10 under the influence of the force in coil spring 111. Ball 122 thus moves out of opening 170 in cap 100 and radially inwards, further into lumen 118. Ball 122 pivots about edge 155 in opening 170 and slides distally along the length of opening 160. When second bevel is aligned with ball 122, it moves as far as it can in opening 160 and is positioned directly above second bevel 72. At that point it will have traveled as far into lumen 117 as it can, constrained by the dimensions of opening 160 and by distal end 101 of cap 100. Spring 111 has expanded and now constrains ball 122 radially. Ball 122 partially occludes lumen 118, thus blocking the passage of needle tip 70 and preventing shield assembly 110 from being pulled back to expose needle tip 70. [0034] At this point, key 190 has reached distal end 270 of slot 260, so further distal movement of shield assembly 110 relative to needle 10 is prevented. The distance from key 190 to needle tip 70 is set so that when tip 70 is aligned with ball 122, there is sufficient space for ball 122 to move beneath cap 100 in opening 160. Upper surface 136 of distal end 101 of cap 100 (i.e. the part of the needle shield assembly 110 that is immediately radially outward of ball 122 and which ball 122 abuts when the shield is deployed) forms an angle a tangential to ball 122 when ball 122 is moving into its position at least partially occluding lumen 118. This can be seen in FIG. 5. This angle α is set at a value less than the smallest bevel angle β of needle tip 70 (bevel 72 in this case). In the described embodiment, the angle α between the upper surface 136 of distal end 101 and ball 122 is about zero degrees. If that angle is made too large relative to angle β, ball 122 will not be trapped. Distal end 158 of stepped area 155 and cap 100 are dimensioned to overhang so that tip 70 can never emerge from shield assembly 110. It is possible to employ multiple balls sitting in multiple openings the same as opening 160 and 170. If this is done, the overhang can be reduced. [0035] After deployment, but before needle 10 moves distally, part of ball 122 lies in lumen 118 and part of it is urged against the inside of distal end 101 of cap 100 by spring 111. The top of ball 122 lies beneath upper surface 136 of distal end 101 of cap 100. In an alternative embodiment, spring 111, having expanded, closes off the opening 170. If needle 10 moves distally, it will abut ball 122, which will be forced against the inside of end 101 of cap 100. Further distal movement of needle 10 and hence emergence of needle tip 70 from shield assembly 110 will be prevented. [0036] Lumen 118 is sized such that needle 10 fits in it snugly. Thus when needle 10 is moved distally (i.e. shield assembly 110 is moved proximally) and ball 122 abuts needle tip 70, needle 10 will not move away from ball 122. Lumen 170 thus provides support opposite ball 122 to prevent needle 10 from wiggling, and to prevent tip 70 from moving such that it pierces the wall of lumen 118. [0037] In an alternative embodiment, ball 122 fully enters lumen 118. Ball 122 thus has a diameter slightly larger than that of lumen 118. Ball 122 is then axially constrained by lumen 118 and needle 10. In this case, lumen 118 is also dimensioned to provide support for needle 10 opposite ball 122, thus preventing wiggle of the needle and preventing tip 70 from piercing the wall of lumen 118. [0038] Ball 122 moves a distance at least equal to the amount by which it protrudes from opening 155 in cap 100. When the shield is deployed, ball 122 extends into lumen 118 by an amount approximately equal to that distance. This leaves part of lumen 118 un-occluded. If a small gauge needle is used a larger ball is needed in order to occlude lumen 118 sufficiently to prevent tip 70 from poking through the un-occluded part of lumen 118 and so that ball 122 will extend from the surface of needle 10 into opening 160. The same effect can be obtained by making cap 100 smaller and using the same sized ball. If a large gauge needle is used (i.e. a needle having large diameter), the ball can be smaller. [0039] Device 5 is assembled in the following way: [0040] 1. Needle shield assembly 110 is dropped into housing 20 from proximal end 210 of housing 20. Key 190 is aligned with slot 260 of housing 20. [0041] 2. Finned member 30 is slid over housing 20, from proximal end 210 of housing 20. During this step, fin 300 is at about 60-90 degrees to the vertical, thus aligning opening 380 or opening 382 with key 190. Finned member 30 is then rotated to the vertical position, locking key 190 behind rim 375. [0042] 3. Spring 180 is placed inside lumen 117 of shield assembly 110, also from proximal end 210 of housing 20. Distal end 190 of spring 180 abuts the back of wall 114 of needle shield. [0043] 4. Needle hub 50 is snapped or glued onto proximal end 210 of housing 20, compressing spring 180. Slot 570 in cylindrical wall of hub 50 is aligned with slot 260 on housing 20. [0044] 5. Needle 10 is threaded into hole 113 of needle shield assembly 110, through lumens 118, 117 and spring 180 and glued into needle hub 55. [0045] 6. Tube 15 is glued into proximal end 555 of needle hub 55. [0046] The device is used in the following way: [0047] The user grasps wings 225 and 230 between his or her fingertips and brings them together so that they touch fin 300. Alternatively, the device can be held simply by grasping fin 300 between the finger tips. Finger grips are provided in fin 300 for that purpose. Holding the device in either of those two ways, with fin 300 in the vertical (first) position, the user pierces the patient's skin and blood vessel with needle tip 70. Once the blood vessel has been pierced and blood can flow through needle 10, the user rotates fin 300 down towards either wing 225 or 230, thus unblocking key 190 (the second position). Needle shield assembly 110 is thus free to slide axially over the needle in the distal direction, urged by spring 180. Fin 300 is placed in cutout 250 or 255, so it is flush with the relevant wing. Wings 225 and 230 and fin 300 can be taped to the patient's skin while the blood is collected. [0048] In this blood collection (second) position, needle shield assembly 110 has slid axially in the distal direction due to the force of spring 180. Distal end 115 of needle shield assembly lies against the patient's skin. As needle tip 70 is withdrawn, distal end 115 of needle shield assembly, still under the influence of spring 180, moves in the distal direction until, as tip 70 is removed from the patient, it is completely shielded. [0049] The shielding mechanism at the tip of the needle prevents needle shield assembly 110 from sliding in the proximal direction and re-exposing needle tip 70. Key 190 abuts distal end 270 of slot 260, thus preventing distal movement of needle shield assembly 110. Needle 10 is thus completely shielded. Even if fin 300 is rotated back into the first position (to facilitate removal of needle 10 from the patient), shield assembly 110 cannot be retracted because it is blocked from proximal movement by ball 122. Key 190 has moved distally with respect to circumferential rim 375. [0050] Although limited embodiments of the winged needle assemblies, their components, and their applications on different needle devices have been specifically described and illustrated, the descriptions are not intended to limit the scope of the basic invention. Many modifications and variations will be apparent to those skilled in the art. Accordingly, it is to be understood that the winged needle assemblies and their components constructed according to principles of this invention may be embodied other than as specifically described herein. The invention is also defined in the following claims. | Summary: A winged needle set is disclosed. The winged needle set has a housing and opposed wings which can be grasped between the fingers for insertion of the needle into a body. The winged needle set has a finned member which is rotatable about the axis of the needle. Rotation of the finned member towards one of the wings actuates a needle shield, which moves along the needle in the distal direction. In one embodiment, the needle shield has includes a blocking object (such as a ball), a biasing spring and a holder for the blocking object. | 6,164 | 128 | big_patent | en |
Summarize: (Reuters) - BlackBerry Ltd (BB.TO) on Tuesday filed a patent infringement lawsuit against Facebook Inc (FB.O) and its WhatsApp and Instagram apps, arguing that they copied technology and features from BlackBerry Messenger. FILE PHOTO - A banner for BlackBerry Ltd hangs to celebrate the company's transfer trading to the New York Stock Exchange (NYSE) in New York, U.S., October 16, 2017. REUTERS/Brendan McDermid Litigation over patent infringement is part of BlackBerry Chief Executive John Chen’s strategy for making money for the company, which has lost market share in the smartphone market it once dominated. “Defendants created mobile messaging applications that co-opt BlackBerry’s innovations, using a number of the innovative security, user interface, and functionality enhancing features,” Canada-based BlackBerry said in a filing with a Los Angeles federal court. “Protecting shareholder assets and intellectual property is the job of every CEO,” BlackBerry spokeswoman Sarah McKinney said in an email. However, she noted that litigation was “not central to BlackBerry’s strategy.” The lawsuit followed years of negotiation and BlackBerry has an obligation to shareholders to pursue appropriate legal remedies, she added. Facebook Deputy General Counsel Paul Grewal said in a statement that the company intended to fight the lawsuit. “Blackberry’s suit sadly reflects the current state of its messaging business,” Grewal said. “Having abandoned its efforts to innovate, Blackberry is now looking to tax the innovation of others.” BlackBerry is trying to persuade other companies to pay licensing royalties to use its trove of more than 40,000 global patents on technology including operating systems, networking infrastructure, acoustics, messaging, automotive subsystems, cybersecurity and wireless communications. BlackBerry is also selling cybersecurity software for self driving cars. BlackBerry sued Nokia Corp in February 2017, alleging infringement of patents relating to 3G and 4G wireless communications technology. That case is still pending in federal court in Delaware. Last year Qualcomm Inc (QCOM.O) agreed to pay BlackBerry $940 million to resolve arbitration over royalty payments. In October 2017 BlackBerry announced a confidential settlement with Blu Products Inc, a Florida-based maker of low-cost mobile devices it had also sued for patent infringement. BlackBerry, the once-great smartphone maker that exited the hardware business in 2016, is suing Facebook for patent infringement. BlackBerry owns a portfolio of broad software patents that cover some of the most basic features of modern smartphone messaging services—and the company says it wants Facebook to pay up. Facebook "created mobile messaging applications that coopt BlackBerry's innovations, using a number of the innovative security, user interface, and functionality-enhancing features that made BlackBerry's products such a critical and commercial success in the first place," BlackBerry's Tuesday lawsuit claims. The lawsuit argues that Facebook subsidiaries Instagram and Whatsapp infringe BlackBerry's patents in addition to Facebook's own messaging apps. It's not unusual for technology companies that lose their lead in the marketplace to turn to patent licensing as an alternative way to make money. Yahoo sued Facebook for patent infringement in 2012, for example, while Nokia sued Apple for patent infringement in 2016. BlackBerry began its own campaign of patent litigation in 2016, suing the little-known Android phone maker BLU and the Internet telephony company Avaya. BLU agreed to pay up last year, and BlackBerry is now moving on to Facebook—potentially a much more lucrative target. BlackBerry is asserting seven software patents against Facebook, and they're remarkably broad: Patent 7,372,961 covers the concept of generating a cryptographic key by choosing a pseudorandom number and then checking if it is "less than order q prior to reducing mod q." If it is, the key is used. If not, another key is chosen at random and the process repeats. Patent 8,209,634 covers the concept of using icons with numeric badges to signal the arrival of new messages. Patent 8,279,173 covers the concept of tagging people in photos using an auto-completing search box. Patent 8,301,713 covers the concept of marking a significant lull in a text message conversation by inserting a timestamp reflecting the time of the next message. Patent 8,429,236 covers the concept of changing how a mobile device sends messages depending on whether they're being actively read by the recipient's device. For example, if updates aren't being read in real time, then the sending device may be able to conserve power by sending messages in batches rather than one at a time. Patent 8,677,250 covers the concept of tying a messaging service and a game application together so that a user playing a game can send messages to contacts on the messaging app that includes updates on the player's progress in the game. Patent 9,349,120 covers the concept of muting a message thread. In short, BlackBerry claims to own some of the most common features of modern mobile messaging apps, whether they're made by Facebook, Apple, Google, or anyone else. BlackBerry has asked the court to enjoin Facebook from infringing these patents, which could require Facebook to dramatically overhaul these apps or even shut them down altogether. But it's unlikely things will get that far. In recent years, the courts have been relatively reluctant to grant injunctions in cases like this, preferring instead to award cash damages that typically wind up being less lucrative to the plaintiff. Facebook may also attempt to get some of these patents thrown out as invalid—the courts have shown skepticism about the legality of some software patents in recent years. We can also expect Facebook to look for patents it can use to countersue BlackBerry, which has a smaller business than it did a few years ago but still sells a significant amount of software and services that could be infringing Facebook patents. And ultimately, there's a good chance that Facebook will sign a patent licensing agreement with BlackBerry—that's how Facebook's dispute with Yahoo turned out, after all. Update: A BlackBerry spokesperson sent us a comment by email: As a cybersecurity and embedded software leader, BlackBerry’s view is that Facebook, Instagram, and WhatsApp could make great partners in our drive toward a securely connected future, and we continue to hold this door open to them. However, we have a strong claim that Facebook has infringed on our intellectual property, and after several years of dialogue, we also have an obligation to our shareholders to pursue appropriate legal remedies. But Facebook signaled that it isn't in a mood to compromise. "Blackberry’s suit sadly reflects the current state of its messaging business," Facebook deputy general counsel Paul Grewal said in an email statement. "Having abandoned its efforts to innovate, Blackberry is now looking to tax the innovation of others. We intend to fight.” | Summary: BlackBerry Limited has long been proud of its messaging app-so much so that it's now suing Facebook and its Instagram and WhatsApp subdivisions for ripping off key features. The Verge notes a "sprawling" 117-page complaint filed Tuesday for patent infringement, alleging that hallmark innovations of BlackBerry Messenger, or BBM-including cryptographic security features, user interface enhancements such as message notifications and photo tagging, and gaming/messaging efficiencies-somehow found their way into the hands of the defendants, which the suit calls "relative latecomers to the mobile messenging world." This theft of intellectual property, the complaint claims, "has succeeded in diverting consumers away from BlackBerry's products and services and toward those of Defendants," which it says "has resulted in a substantial and undeserved windfall for Defendants." Ars Technica, which details BlackBerry's "remarkably broad" patents, adds the company got out of making hardware in 2016; its smartphones are now made by Chinese firm TCL, while it focuses on mobile security and software products. Bloomberg notes the suit "might seem strange," as phone messaging apps are now "ubiquitous" and share many of the same features. A BlackBerry rep tells Reuters the suit came only after longtime negotiations and because the company owed it to shareholders to protect its technology. Cheddar reporter Alex Heath, who initially said BlackBerry's suit felt "desperate and completely out of left field," tweeted out a statement Tuesday from Facebook, which notes "Blackberry's suit sadly reflects the current state of its messaging business. Having abandoned its efforts to innovate, Blackberry is now looking to tax the innovation of others. We intend to fight." | 1,574 | 382 | multi_news | en |
Write a title and summarize: Humans and other species have perceptual mechanisms dedicated to estimating approximate quantity: a sense of number. Here we show a clear interaction between self-produced actions and the perceived numerosity of subsequent visual stimuli. A short period of rapid finger-tapping (without sensory feedback) caused subjects to underestimate the number of visual stimuli presented near the tapping region; and a period of slow tapping caused overestimation. The distortions occurred both for stimuli presented sequentially (series of flashes) and simultaneously (clouds of dots); both for magnitude estimation and forced-choice comparison. The adaptation was spatially selective, primarily in external, real-world coordinates. Our results sit well with studies reporting links between perception and action, showing that vision and action share mechanisms that encode numbers: a generalized number sense, which estimates the number of self-generated as well as external events. Animals, including humans, estimate spontaneously and reasonably accurately the approximate quantity of arrays of objects, without recourse to other forms of representation, such as density (Cicchini et al., 2016). Even newborn infants of less than 3 days show selective habituation to number (Izard et al., 2009). There is now very good evidence in both human and non-human primates that number is encoded by intraparietal and prefrontal cortex (Castelli et al., 2006; Dehaene et al., 2003; Harvey et al., 2013; Nieder, 2005,2012,2016; Nieder et al., 2006; Nieder and Miller, 2004; Piazza and Eger, 2016; Piazza et al., 2004,2007), even in numerically naive monkeys (Viswanathan and Nieder, 2013). All these studies point to the existence of a visual sense of number within a parietal–frontal network (Dehaene, 2011). A truly abstract sense of number should be capable of encoding the numerosity of any set of discrete elements, displayed simultaneously or sequentially, in whatever sensory modality. Some evidence exists for such a generalized number sense. Neurons in the lateral prefrontal cortex (lPFC) of behaving monkeys encode numerosity for both auditory and visual sensory modalities, suggesting supra-modal numerosity processing (Nieder, 2012). Another study reported separate populations of neurons in the intraparietal sulcus (IPS) responding selectively to sequential or simultaneous numerical displays, while a third set of neurons showed numerosity selectivity for both simultaneous and sequential presentations, suggesting that the information about spatial and temporal numerosity converges to a more abstract representation (Nieder et al., 2006). There is also evidence from functional imaging in humans for a right lateralized fronto-parietal circuit activated by both auditory and visual number sequences, and that right IPS is involved in processing both sequential and simultaneous numerosity formats (Castelli et al., 2006; Piazza et al., 2006). Psychophysical evidence showing little cost in cross-modal or cross-format matching also points to a common number sense spanning sensory modalities and formats. For example, human adults are very efficient in making cross-modal and cross-format judgments, with very little cost in either accuracy or reaction times when comparing auditory with visual temporal sequences or dot arrays (Barth et al., 2003; Brannon, 2003). Developmental work also show similar accuracy in pre-schoolers for comparing spatial array of dots either with other spatial arrays, or with sequences of sounds (Barth et al., 2005). Preferential-looking studies show infants prefer to look at screens displaying adults faces numerically matched with the soundtrack of adult voices (Jordan and Brannon, 2006), and abstract visual ensembles (shapes) numerically matched with on-going sequence of sounds (Izard et al., 2009). However, not all agree: Tokita & Ishiguchi (Tokita and Ishiguchi, 2012) reported significantly lower precision for cross-format number comparisons in adults, than for within format comparisons. That there is little or no cost in these matches is certainly indicative of efficient transfer of information between senses, but says little about the mechanisms involved. The match is made at the decision level, so the interaction could be at any stage up to and including decision mechanisms. One of the more powerful psychophysical techniques to probe mechanisms is adaptation (Mollon, 1974; Thompson and Burr, 2009). Number, like most other primary visual attributes, is also highly susceptible to adaptation (Schwiedrzik et al., 2016; Burr and Ross, 2008): visually inspecting for a few seconds a large number of items results in the perceived numerosity of a subsequent ensemble to be strongly underestimated, and vice-versa after adaptation to low numbers (Burr and Ross, 2008). More recently we have shown that adaptation to numerosity also occurs with sequentially presented stimuli, and that the adaptation effects are both cross-modal and cross-format (Arrighi et al., 2014): adapting to sequences of tones affects the perceived numerosity of a subsequently presented series of flashes (and vice versa), and adapting to sequences of flashes affects the perceived numerosity of spatial arrays of items. Importantly, the adaptation was spatially selective, in external rather than eye-centered coordinates, suggesting it has a perceptual rather than cognitive basis. fMRI studies have demonstrated BOLD activity selective for adaptation to numerosity in the human parietal sulcus (Piazza et al., 2004,2007; Castaldi et al., 2014; He et al., 2015). All these studies strongly suggest that the number sense is a high-generalized system, capable of combining numerical information from different senses, and across different formats. Numerical information is also relevant for the production of specific action sequences, from dance routines to more simple repetitive behavioral tasks. A few studies point to an interconnection between numerosity and motor control. For example, neurons in area 5 of the superior parietal lobule of monkey show a clear selectivity for the number of self-produced actions, and inactivation of the area impedes number-based tasks (Sawamura et al., 2002,2010). Other work has shown that the left ventral premotor cortex is activated by counting successive sensory stimuli (Kansaku et al., 2007), and that the human cerebellum shows strong activation for simple numerical calculations (Arsalidou and Taylor, 2011). The existence of anatomical and functional connections between number and action-generation systems raise the possibility that number-for-action could be encoded within a truly abstract numerosity mechanism. To test this idea, we measured cross-adaptation between motor repetitions and perception of numerosity. The results show that adapting to self-generated action does affect the representations of numerosity of external events, both sequential (series of flashes) and simultaneous (dots ensembles), and that the adaptation is spatially selective in external, not hand-centered coordinates. Each trial began with a motor adaptation phase in which participants performed tapping movements for six seconds, under two different conditions (tested on separate sessions):' high adaptation', where participants were asked to tap as quickly as possible (average 5–6 taps/s); and' low adaptation' where they tapped more slowly (average 1. 12 taps/s: see Materials and methods and Figure 5). After the adaptation phase, the test stimulus – either a sequence of flashes or a cloud of dots (tested on separate sessions) – was randomly displayed either to the same side of the screen where the hand had been tapping, or to the symmetrically opposite side. Participants estimated the numerosity of the test stimulus, which varied randomly from trial to trial within the range 6–14. To minimize sensory feedback, participants were placed in a dark room and wore soundproof headphones, and tapped in mid-air behind the computer screen without touching any surface. The results are shown in Figure 1. Panels A & B show numerosity estimates averaged over all subjects as a function of the physical numerosities displayed. When the test stimulus was displayed on the right side of the screen (where the adaptation had occurred), rapid tapping caused a consistent underestimation of the numerosity of the test, while slow tapping caused an overestimation. The adaptation effects were similarly strong for when the test was a sequence of flashes (Figure 1A) as when it was an array of dots presented simultaneously (Figure 1B). Interestingly, the effect occurred only when the stimuli were presented on the same side as the tapping hand (the right side): when presented on the other (left) side, adaptation produced no consistent effect (Figure 1A and B open symbols). 10. 7554/eLife. 16161. 003Figure 1. Effects of motor adaptation on perceived numerosity. (A and B) Average perceived numerosity as a function of physical numerosity for slow tapping (downward triangles) and fast tapping (upward triangles), for sequential (left) and simultaneous (right) formats. Filled symbols indicate the conditions in which stimuli were spatially congruent with the tapping region, small open symbols to estimates obtained for the unadapted location (left-hand side). (C and D). Adaptation magnitudes for individual subjects when test and tapping were spatially congruent, plotted against the spatially incongruent condition. Stars reports averages, squares single subject data. Error bars refer to ± 1 SEM. DOI: http: //dx. doi. org/10. 7554/eLife. 16161. 003 We defined adaptation magnitude as the percentage difference in perceived numerosity after adaptation to fast or slow tapping, averaged over all numerosities. For sequential and simultaneous presentations, the adaptation magnitude averaged across subjects (filled symbols in Figure 1C and D) was around 20 and 25% respectively for stimuli presented to the adapted location, a very strong effect. For stimuli presented to the unadapted location, the average effect was only 4 & 2%. We also calculated adaptation magnitude for individual subjects. Figure 1C and D plot adaptation magnitudes for the congruent condition (where the visual stimuli were presented to the right side), against the incongruent condition (stimuli to the left side). All subjects showed a significant effect in the congruent condition (error bars 1 sem), but very little effect in the incongruent condition. ANOVA showed that the congruent conditions were highly significant (F (1,32) = 70. 219, p = 0. 001, η2 = 0. 29, Cohen’s d = 1. 278 and F (1,48) = 47. 176, p = 0. 0004, η2 = 0. 217, Cohen’s d = 1. 062 for sequential and simultaneous condition respectively), while the non-congruent conditions were weak and insignificant (≈ 4% effect, F (1,32) = 1. 403, p = 0. 302, η2 = 0. 007, Cohen’s d = 0. 167 and ≈ 2% effect, F (1,48) = 0. 919, p = 0. 375, η2 = 0. 008, Cohen’s d = 0. 179). That the adaptation is spatially specific suggests it is of a perceptual rather than cognitive nature, and unlikely to result from a response bias or any other generalized artifact. This first experiment revealed two clear results: that motor adaptation affects visual estimates of numerosity, for both sequential and simultaneous displays; and that the adaptation is spatially specific. The spatial specificity suggests that the effect is not a high-level, cognitive phenomenon (such as' internal counting' ), but perceptual in nature, mediated by neural mechanisms with circumscribed receptive fields. To verify the robustness of the spatial selectivity, and to understand it better, we repeated the experiment with a new subject pool, changing the tapping hand and location. In this experiment we tested only the simultaneous presentation, as this is the most revealing (and surprising) result. The violet symbols of Figure 2A replicate the results of the previous experiment, tapping with the right (dominant) hand and testing on both right and left sides (randomly interleaved): the adaptation effect was again strong for stimuli presented on the same side (filled symbols), and non-existent for stimuli on the other side (open symbols) (F (1,40) = 70. 207, p = 0. 000397, η2 = 0. 116, Cohen’s d = 0. 724; F (1,40) = 2. 036, p=0. 213, η2 = 0. 0019, Cohen’s d = 0. 0873; for adapted and unadapted location respectively). The red symbols of Figure 2B show the results for tapping on the left with the left (non-dominant) hand: again the effects occurred only for visual stimuli presented on the congruent side (left), although they were somewhat weaker (F (1,40) = 9. 305, p = 0. 028, η2 = 0. 05, Cohen’s d = 0. 4588; F (1,40) = 0. 265, p = 0. 629, η2 = 0. 001, Cohen’s d = 0. 0633; for adapted and unadapted location respectively). Figure 2C shows results for tapping with the dominant (right) hand on the left side of the screen. Here, adaptation was found only for stimuli presented to the left side of the screen, suggesting that it is spatially selective in external rather than hand-centered coordinates (F (1,40) = 36. 840, p = 0. 002, η2 = 0. 104, Cohen’s d = 0. 6814; F (1,40) = 1. 380, p = 0. 293, η2 = 0. 0023, Cohen’s d = 0. 096; for adapted and unadapted location respectively). Figure 2D shows the results for all six subjects. There is some variability between subjects, particular in the crossed condition, where one subject showed adaptation to stimuli on the right after tapping on the left with the right hand, but by and large the individual data reinforce the group data. 10. 7554/eLife. 16161. 004Figure 2. Reference frame of motor adaptation. (A) Average perceived numerosity as a function of physical numerosity for the slow-and fast-tapping conditions (downward and upward triangles respectively), for right-hand tapping. Filled symbols refer to trials when the stimuli were presented in the spatial region where the subjects had tapped (right side) small open symbols to trials when the stimuli were presented on the other side. This data replicates Figure 1B with a fresh subject pool. (B) Same as A, except subjects tapped with their left hands. Filled symbols refer to testing in the same spatial region where the subjects had tapped (left side), small open symbols to the right side. Other conventions like A. (C) Same as A, except the right hand tapped on the left side of the screen. Filled symbols refer to testing on the same spatial region where the subjects had tapped (left side), small open symbols to the right side. (D) Adaptation magnitudes for individual subjects when test and tapping were spatially congruent, plotted against the spatially incongruent condition. Color-coding as for A, B and C (purple: right hand, right side; red: left hand, left side; orange: right hand, left side). Stars reports averages, squares single subject data. Error bars refer to ± 1 SEM. DOI: http: //dx. doi. org/10. 7554/eLife. 16161. 004 In the previous experiment, subjects tapped in mid air to minimize sensory feedback. In the next series of experiments we manipulated the amount of sensory feedback in the adaptation phase to examine interactions between sensory and motor signals. In the first condition (tactile only), subjects tapped a mouse behind the monitor, allowing for tactile feedback (Figure 3A). The adaptation effect in this condition was strong, around 20% (F (1,40) = 743. 738, p = 0. 0001, η2 = 0. 203, Cohen’s d = 1. 009). In the next condition (visual and tactile), the monitor accompanied each mouse-tap with a flash, to give visual as well as tactile feedback. Despite the extra feedback, adaptation remained around 20%, (F (1,40) = 36. 746, p = 0. 002, η2 = 0. 184, Cohen’s d = 0. 949) as shown in panel B of Figure 3. The last adaptation condition (visual only) comprised a sequence of visual flashes whose rates were determined by the adapting motor routine of the previous conditions (visual and tactile). Again, the adaptation effect was found to be strong (F (1,40) = 61. 740, p = 0. 001, η2 = 0. 230, Cohen’s d = 1. 093), and similar to the other conditions, around 20% (Figure 3C), making these three adaptation conditions equally effective as tapping in mid-air (F (4,29) = 0. 475, p = 0. 754, η2 = 0. 07, Cohen’s d = 0. 548: see Figure 3D). 10. 7554/eLife. 16161. 005Figure 3. Role of sensory feedback of motor adaptation on perceived numerosity. (A), (B), (C) Average responses as a function of physical numerosity for slow adaptation (downward triangles), fast adaptation (upward triangles) and no adaptation (diamonds), for the three different conditions. (D) Bar graphs report the average adaptation effect for all adapting conditions (tactile only - red; visual and tactile - blue, visual only – black and the 2 conditions of Exp 1: sequential-green and simultaneous-violet). Open symbols show single subject data. Error bars report ± 1 SEM. All the conditions provided significant effects (all p-values < 0. 05). The magnitude of the effect does not differ between conditions (p > 0. 05). DOI: http: //dx. doi. org/10. 7554/eLife. 16161. 005 We also verified the results with a two-alternative forced-choice technique. Subjects adapted to high or low tapping rates, as in the first experiment (no tactile or visual feedback), then two clouds of dots were simultaneously presented to the right (adapted) and left (unadapted) positions. The numerosity of each stimulus varied from trial to trial over the range 5–20, and subjects indicated which stimulus appeared more numerous. Figure 4A plots average responses as a function of the difference between the right and the left stimulus (normalized to the average of the two numerosities), to yield psychometric functions. The effect of adaptation was again clear: adapting to low tapping rates shifts the curve to the left (compared with baseline), consistent with an overestimation of the perceived numerosity (t (5) = 3. 285, p = 0. 021, Cohen’s d = 1. 101) and high tapping rates caused the opposite effect, even if weaker (t (5) = 1. 237, p = 0. 27, Cohen’s d = 0. 558). The differences in the points of subjective equality (PSEs, given by the 50% point of the curves) of the two adapting conditions again gives an index of magnitude of adaptation, around 15%. Figure 4B shows the PSEs for adaptation to the two conditions. Despite some variability amongst subjects the effects are quite robust and statistical significant as shown by a two-tailed paired t-test: t (5) = 3. 56, p = 0. 029, Cohen’s d = 1. 612. This experiment confirms the main results with a different technique, and also confirms the spatial selectivity of the adaptation: if adaptation was not spatially selective, it would work equally on the presentations to the left and right sides, annulling the effect. 10. 7554/eLife. 16161. 006Figure 4. Forced-choice measurement of motor adaptation. (A) Psychophysical functions for pooled data (6 subjects) after adaptation to fast (light violet circles), slow (dark violet triangles) or no (black squares) tapping. The curves indicate the proportion of trials when the test (presented on the right, the same side of tapping) was seen as more numerous than the unadapted stimulus (presented on the left), as a function of the numerosity difference (normalized by the averaged of the two stimuli). Adaptation to slow tapping shifted the curve leftwards, showing that subjects were biased to perceive the stimulus as more numerous that it was; and adaptation to fast tapping shifted it rightwards. The point where the best-fitting curves pass 50% is considered the point of subjective equality (PSE, indicated by the coloured arrows). (B) PSEs for individual subjects after adaptation to fast tapping (ordinate) against those after adaptation to low motor repetitions (abscissa). The filled star shows results for data averaged across subjects. Error bars report ± 1 SEM. DOI: http: //dx. doi. org/10. 7554/eLife. 16161. 006 This study shows that estimates of numerosity, both sequential and simultaneous, are strongly biased after adapting to repetitive finger tapping: rapid tapping decreases apparent numerosity, slow tapping increases it. The effect is spatially selective, primarily in external rather than hand-centered coordinates. There has been a long-standing debate as to whether adaptation effects operate on numerosity per se, or via texture-density mechanisms (Burr and Ross, 2008; Anobile et al., 2014,2015,2016; Bell et al., 2015; Dakin et al., 2011; Durgin, 1995,2008; Morgan et al., 2014; Ross and Burr, 2012; Ross, 2010; Tibber et al., 2012,2013). A similar argument could be made here: that the adaptation was to temporal frequency, rather than to numerosity. As with spatial adaptation, there are many reasons to suggest that this is unlikely. However, the cross-format adaptation (adapt to tapping sequence and test on dot array) clearly rules out this possibility: the spatial arrays are not temporally modulated. It is numerosity that is being adapted, not temporal frequency. The current results reinforce the many previous studies (Izard et al., 2009; Nieder, 2012; Nieder et al., 2006; Barth et al., 2003; Brannon, 2003; Barth et al., 2005; Jordan and Brannon, 2006; Arrighi et al., 2014; Jordan et al., 2005) discussed in the introduction that point to the existence of a generalized sense of number. Most of these studies relied principally on cross-modal comparisons of number, which could occur at any processing stage, up to and including decision mechanisms. The spatial selectivity shown in our study suggests that the interaction is perceptual rather than cognitive: adapting on the left side did not affect stimuli on the right, and vice versa. Importantly, the specificity was in external coordinates, as adapting the left field with the right hand caused adaptation for visual stimuli presented to the left, not the right visual field. This complements nicely the result of our previous study (Arrighi et al., 2014), where we showed that adaptation to visual sequences affects number perception of both sequential and simultaneous presentations, in a spatially selective manner. Interspersing an eye-movement between adaptation and test showed that the adaptation was spatially specific in external rather than eye-centered coordinates: as the current study shows the selectivity is external, not hand-centered. It would be interesting to look at the spatial tuning of the adaptation on a finer grain, to define the size of the adaptation field. The present study shows that the adaptation is at least broadly tuned, confined to a particular hemifield. It would be very informative to determine whether there was also selectivity within each hemifield, and on how fine a grain. Some may find the spatial selectivity of the adaptation difficult to reconcile with the concept of a generalized, abstract sense of number. However, cross-modal effects can also show spatial selectivity. For example, cross-modal integration of visual and auditory (or tactile) information occurs only if the stimuli are spatially coincident (within certain bounds) (Slutsky and Recanzone, 2001). Similarly event time, which certainly transcends modalities, and also seems to be coded in parietal cortex (Leon and Shadlen, 2003), is affected by motion adaptation, in a spatially selective manner (Burr et al., 2007; Fornaciai et al., 2016). Interestingly, the spatial selectivity of the adaptation is in external – not eye-based – coordinates as we observed for number, here and in the previous study (Arrighi et al., 2014). We tested adaptation to action under various feedback conditions: visual and tactile, only visual, only tactile, and minimal feedback. All conditions produced similar amounts of adaptation. In the' minimal feedback' conditions, where subjects tapped in mid-air, there was no tactile feedback from hitting a surface. We could not, however, remove all forms of kinaesthetic feedback, and therefore cannot be certain whether the adaptation signal was the intension to move, or the sensory proprioceptive feedback from the finger. But both are signals about action, whether they are' inflow' or' outflow'. It is interesting that this condition with reduced perceptual feedback produced the same amount of adaptation, as did the conditions with visual and/or tactile feedback. It is also interesting that the vision-only condition produced similar adaptation. Many studies have suggested that vision and action are linked (Arrighi et al., 2011; Goodale and Milner, 1992). This study is a further clear example of their interconnection, in the encoding the numerosity of internally generated actions and externally generated events. A total of 15 adults (13 naïve to the purpose of the study, 2 author; mean age 27, all right-handed with normal or corrected-to-normal vision) participated in the numerosity estimation experiments. Six of them were tested in the sequential condition (test stimuli: sequences of flashes) and 7 of them in the' simultaneous condition' in which test stimuli consisted of array of dots simultaneously presented. Three of these (2 author and 1 naïve subject) also participated, together with 3 additional naïve subjects (mean age of group: 28), in the second experiment investigating the reference frame of the motor adaptation after-effect. Eventually, six subjects (mean age of group: 28) were tested in the experiment concerning forced-choice discrimination of numerosity. All participants gave written informed consent. Experimental procedures were approved by the local ethics committee (Comitato Etico Pediatrico Regionale—Azienda Ospedaliero-Universitaria Meyer—Firenze FI) and are in line with the declaration of Helsinki. Stimuli were created and presented with Psychophysics toolbox for Matlab and displayed on a 60 Hz - 17”, touch screen monitor (LG-FLATRON L1732P) placed at a subjects view distance of 57 cm. To eliminate auditory feedback, participants wore soundproof headphones. In some conditions, hand movements were monitored by an infrared motion sensor device (Leap motion controller - https: //www. leapmotion. com/) running at 60 Hz. During the design of the experiments we computed an appropriate sample size to confidently report an effect of motion adaptation on perceived numerosity. Sample size was measured by means of a one-sample t-test assuming a value of 0 (no effect) as a Null Mean and retrieving a value for alternative mean and standard deviation from a previous study of our group (see Figures 4 and 5 in Arrighi, et al. [Arrighi et al., 2014]). The analysis revealed that with a sample size of 4, a power of 0. 95 was achieved with an alpha level of 0. 01. For this reason in all or experiments, we always tested a number of participants greater than 4 (see below for details). We did not set any inclusion criteria for subject selection or their data: all data, for all experimental conditions, were analyzed and reported. In all conditions where subjects estimated numerosity we tested statistical significance with a 2 × 9 repeated measures ANOVA with test numerosity (9 levels for numerosity, range 6–14) and adaptation type (low and high) as main factors. Difference in the adaptation effects between the several adaptation conditions (visual, tactile, visual-tactile, and the two conditions with minimal feedback) were measured by a one-way ANOVA. In the numerosity discrimination task, difference in the adaptation effects for high and low adaptation were tested for statistical significance by mean of two-tailed paired t-test. For t-test analyses we measured Cohen' s d. For repeated measures ANOVA and regression analyses, we reported both Cohen' s d and η2. Here Cohen' s d was measured transforming η2 into Cohen' s d (Cohen, 1988). All data are publicly available at Figshare (Anobile et al., 2016). Figure 5 plots the tapping rate for the fast against the slow adaptation conditions, expressed as actions per second (Hz). Different colors and symbols refer to different experimental conditions (see caption). On average (across trials and conditions), when asked to tap quickly, participants tapped at a frequency of 5–6 Hz (for a total number of 30–36 tapping repetitions) with almost no difference between the adapting conditions: mean 5. 33 ± 0. 9; 5. 48 ± 0. 5; 5. 2 ± 0. 7; 5. 54 ± 0. 8; 6. 19 ± 0. 37; 5. 69 ± 0. 38; 5. 67 ± 0. 31 for the ‘sequential’, ‘simultaneous’, ‘visual and tactile’, ‘tactile only’, ‘adapt with the right hand in the right space’, ‘adapt with the left hand in the left space’ and ‘adapt with the right hand in the left space’ respectively. Also tapping frequencies for the condition in which subjects tapped slowly were similar across adapting conditions with all values ranging between 0. 7 and 1. 3 Hz (mean 1. 31 ± 0. 4; 1. 29 ± 0. 3; 1. 12 ± 0. 4; 0. 7 ± 0. 3; 1. 18 ± 0. 12; 1. 07 ± 0. 13; 1. 18 ± 0. 17 for the ‘sequential’, ‘simultaneous’, ‘visual and tactile’, ‘tactile only’, ‘adapt with the right hand in the right space’, ‘adapt with the left hand in the left space’ and ‘adapt with the right hand in the left space’ respectively). These data clearly indicate that regardless the tapping routine to be performed on a rigid surface or in mid-air, the tapping temporal dynamics were always very similar. We also tested whether there was a correlation between faster tapping rate and adaptation effects. There was a slight, but non-significant tendency for faster tapping rates to be associated with lower adaptation. But as the correlation was not significant, we assume that variable tapping rates was not a cause for concern for the results of these experiments. | Title: A shared numerical representation for action and perception Summary: Humans and many other animals have the ability to make spontaneous and rapid estimates of the approximate number of items that they can see. This sense of number, or "numbersense", is particularly important in humans, as evidence suggests that it lays the groundwork for acquiring mathematical skills. Researchers have many questions about numbersense. Is it a kind of perception? Or does it require more active thought, like counting? Do people have the same sense of number when they view, hear or touch items that depict the same number? Having a sense of number is essential for carrying out certain actions, like the following the steps in a dance, but the connection between action and numbersense is not entirely clear. A process called adaptation means that viewing specific stimuli for a period of time can affect what people think they see subsequently. For example, viewing large numbers of dots makes subsequent smaller groups of dots seem like they contain fewer dots than they actually do. Anobile, Arrighi et al. have now investigated the link between action and numbersense by asking volunteers to tap one hand either rapidly or slowly in one spot for a short time. The volunteers were then shown a series flashes or a cloud of dots in the region where they had been tapping and asked to estimate the number of flashes or dots. After fast tapping, the volunteers greatly underestimated the numbers of flashes or dots that they saw; after slow tapping, they overestimated the numbers. However, if the images were shown far away from where the volunteers had been tapping, their estimates were more accurate. Overall, the results suggest that adaptation is controlled by space-specific sensory mechanisms rather than some kind of active counting. Furthermore, numbersense appears to have a generalized form that is shared by the brain regions responsible for perception and action. Because numbersense and mathematical ability are linked, this strong connection between action and number perception may have important implications for understanding and treating math-related learning disabilities. Anobile, Arrighi et al. next plan to study how movement-driven adaptation affects numbersense in children and adults with these conditions. | 7,795 | 457 | lay_elife | en |
Write a title and summarize: Identifying master regulators of biological processes and mapping their downstream gene networks are key challenges in systems biology. We developed a computational method, called iRegulon, to reverse-engineer the transcriptional regulatory network underlying a co-expressed gene set using cis-regulatory sequence analysis. iRegulon implements a genome-wide ranking-and-recovery approach to detect enriched transcription factor motifs and their optimal sets of direct targets. We increase the accuracy of network inference by using very large motif collections of up to ten thousand position weight matrices collected from various species, and linking these to candidate human TFs via a motif2TF procedure. We validate iRegulon on gene sets derived from ENCODE ChIP-seq data with increasing levels of noise, and we compare iRegulon with existing motif discovery methods. Next, we use iRegulon on more challenging types of gene lists, including microRNA target sets, protein-protein interaction networks, and genetic perturbation data. In particular, we over-activate p53 in breast cancer cells, followed by RNA-seq and ChIP-seq, and could identify an extensive up-regulated network controlled directly by p53. Similarly we map a repressive network with no indication of direct p53 regulation but rather an indirect effect via E2F and NFY. Finally, we generalize our computational framework to include regulatory tracks such as ChIP-seq data and show how motif and track discovery can be combined to map functional regulatory interactions among co-expressed genes. iRegulon is available as a Cytoscape plugin from http: //iregulon. aertslab. org. Precise regulation of gene expression is imperative for all biological processes. Sequence-specific transcription factors (TFs) bind to their DNA recognition sites within cis-regulatory elements and thereby contribute to the control of the transcriptional initiation rate of their target genes through an interplay with other transcription factors, co-factors, chromatin modifiers, and transcription factories [1]–[3]. The human genome encodes for about 1800 sequence-specific TFs, each of which regulates hundreds of target genes [1], [4], [5]. Because TFs play key roles in gene expression, they are often considered the master regulators of cellular processes. Thus, the mapping and characterization of their regulon (all the target genes of a TF) can provide crucial insight into the biological processes they control [6], [7]. For example, in cancer, ∼40% of the driver mutations affect TFs, and many of the key oncogenes and tumor suppressors, such as p53, MYC, E2F, and NF-κB, are transcription factors [8]. Identification of the TFs that operate a perturbed gene network, and detecting their target genes, are instrumental steps in uncovering key insights into oncogenic programs, including the discovery of therapeutic targets [9]–[12]. For example, although many target genes have been described for the tumor suppressor p53 [9], [13], [14], several aspects of the gene regulatory network (GRN) downstream of p53 remain unknown. For example, it is still unclear whether p53 also directly represses target genes; whether p53 cooperatively regulates target genes with particular co-factors; and whether different target genes are regulated depending on the cancer type, or depending on the context of p53 activation. The situation is obviously worse for less studied TFs for which often none or only few target genes are known. The targets of a known TF can be identified experimentally with relatively high accuracy through chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) [15]. However, ChIP-Seq has limitations because it is usually applied to cells in culture rather than to the actual biological sample (e. g., a tumor); and it focuses on a single TF at a time, that has to be chosen a priori. When the TF is not known in advance, or when only gene expression profiling can be performed, regulatory relationships can be uncovered by reverse-engineering a gene regulatory network starting from the expression data. One approach to solve this problem is by exploiting the fact that genes that are co-regulated by the same TF commonly share binding sites for this TF. However, detecting these short and variable TF binding sites (TFBS) within large non-coding regions represents a computational challenge when working with human or mouse genomes. Although a lot of progress has been made over the last decade and many motif discovery methods have been developed and refined (reviewed in [16]–[19]), motif discovery methods alone are not sufficient to map a gene regulatory network, nor can they be applied to noisy gene sets containing mixtures of targets of multiple TFs. This is true for both motif discovery methods relying on de novo detection and those relying on the enrichment of known position weight matrices (PWM). Additionally, many tools have a motif-oriented output, making it difficult to identify the possible upstream TF. A further limiting factor is that many methods are restricted to using human annotated PWMs (e. g. TRANSFAC [20], JASPAR [21] or UNIPROBE [22]), limiting the number of TFs that can be identified as candidate network regulators based on motif enrichment. Therefore, although cis-regulatory sequence analysis has great potential in resolving direct TF-target interactions, it has until today seen limited applications towards gene regulatory network mapping. Finally, the recent availability of thousands of ChIP-Seq datasets, both from ENCODE [23], and other resources [24], yields new opportunities to discover master regulators from co-expressed gene sets [25], while at the same time pose challenges on how to integrate these data with motif discovery. Here, we aim to tackle some of these challenges by increasing the performance of motif detection to yield high-confidence results, even in noisy gene sets. Motif detection is followed by the annotation of the discovered motifs with associated TFs and direct targets. To this end, we have collected more than nine thousand PWMs from various sources and from different species and link them to candidate binding TFs using a “motif2TF” procedure. This will allow the user to link hitherto anonymous motifs, and motifs of TFs from other species, to candidate human TFs. Furthermore we developed a user-friendly Cytoscape plugin [26], called iRegulon, allowing the integration of predicted cis-regulatory binding sites directly into a biological network. Finally, we extend and generalize this framework towards combined motif and track discovery on a co-expressed gene set, incorporating more than 1000 ChIP-Seq tracks. The iRegulon Cytoscape plugin is available via the Cytoscape App Store [27] and can be downloaded from http: //iregulon. aertslab. org/. The goal of iRegulon is to enable gene regulatory network mapping directly based on motif enrichment in a co-expressed gene set. As motif discovery method we have chosen to elaborate on the recent ranking-and-recovery methods [28]–[32] (Fig. 1). In the ranking step we generate whole-genome rankings of 22284 human RefSeq genes for a library of PWMs where a PWM is a matrix representation of a regulatory motif (Table 1). For each gene, a regulatory search space (500 bp, 10 kb or 20 kb around the Transcription Start Site (TSS), see Materials and Methods) is scanned for homotypic cis-regulatory modules (CRM) using a Hidden Markov Model [33] (Fig. S1). Starting from a library with N PWMs, N ranked lists of genes are generated, each with the most likely genomic targets of a particular motif at the top of the ranking [28], [29]. Next, orthologous search spaces in ten other vertebrate genomes are determined by UCSC liftover tool [34] and are subsequently scanned with the same PWMs. The rankings for different species are combined by rank aggregation [35] into one final ranking for each PWM in our library. For the PWM libraries we have collected and reformatted most of the available libraries into a “6K collection” (N = 6383 PWMs) and a “10K collection” (N = 9713 PWMs) (Table 1). These libraries contain PWMs from different species and also include candidate PWMs for unknown TFs. The results of the ranking step are N human gene rankings stored in an SQLite database. We also generated similar databases using mouse and Drosophila as reference species, in case the input gene set is derived from mouse or fruit fly. The recovery step uses as input any set of co-expressed genes (Fig. 1B). The enrichment of these genes is determined in each of the N motif-based rankings using the Area Under the cumulative Recovery Curve (AUC), whereby the AUC is computed in the top of the ranking (default set to 3%, see Fig. S2 for validation). The AUC values are normalized into a Normalized Enrichment Score (NES) on which we set a default cutoff of 3. 0, corresponding to a False Discovery Rate (FDR) between 3% and 9% (Fig. S3 and Materials and Methods). The leading edge of candidate targets is selected as the optimal subset of highly ranked genes compared to the genomic background and compared to the entire motif collection as background (Fig. 1B and Materials and Methods). We have previously successfully applied the ranking-and-recovery method for Drosophila, namely in cisTargetX [29] and i-cisTarget [28]. These methods have been proven successful in identifying upstream regulators and direct target genes from co-expressed gene sets for Atonal [29], Shavenbaby [36], Fruitless [37], EcR [38], Dichaete [39], Glass [40], dJun/Vri [41], and Rfx [42]. Here, we apply this framework for the first time to human and mouse and we add two novelties to facilitate GRN mapping. The first is a motif2TF procedure that links an enriched motif (PWM) to a candidate binding TF (Fig. 1C and Materials and Methods). For this step we constructed a database of motif-TF direct annotations, TF-TF edges as defined by gene homology [43], [44], and motif-motif edges as defined by motif similarity (using Tomtom [45]). The database links 6031 motifs from the “10K” collection to 1191 human TFs. The advantage of this method is that it allows discovery of motif-TF links based on orthology and based on similarities between annotated and “unknown” motifs in the collection. Application of this method adds 247 more TFs to be identified than the 944 directly annotated TFs in human, and vastly increases the number of different motifs per TF (see Materials and Methods for more detailed description). The second novelty is the availability of the method as a Cytoscape [26] plugin, called iRegulon. The plugin works on any input network and returns a combination of regulators, their direct targets within the input network, and their binding motifs. A detailed description on the use of the plugin is provided in Fig. S4. This is, to our knowledge, the first method that brings cis-regulatory sequence analysis into Cytoscape. This dramatically changes the way motif discovery is performed, because instead of a list of promoter sequences used as input, now any set, network, or pathway of genes can be used as input. Instead of a list of enriched motifs, regulons, are the output, containing the candidate TFs along with their optimal direct target subsets. iRegulon results can be immediately used to map (or annotate) gene regulatory networks and be integrated with the extensive array of regulatory, expression, and annotation tools available within Cytoscape. To evaluate the performance of iRegulon, we derived direct target gene sets for 115 sequence-specific TFs from the ENCODE ChIP-Seq data [46], and for each target set we investigate whether the ChIP' ped TF can be correctly recovered (see Materials and Methods). Out of 115 tested TFs, iRegulon correctly identifies up to 94 TFs (82. 6%) with Normalized Enrichment Scores (NES) above 3 (Fig. 2A, and Materials and Methods). We found iRegulon to be robust to noisy gene sets by adding increasing levels of noise (negative genes) to each set of targets (Fig. 2B). The motif2TF step is crucial to link an enriched motif to a candidate TF; and including motifs from other species and unknown motifs allow detecting many more correct regulators compared to using only known human motifs from TRANSFAC or JASPAR (Fig. 2C). After optimizing the parameters of iRegulon and motif2TF (see Materials and Methods and Fig. S2), we compared iRegulon with eight other motif discovery methods that use a similar input (a set of co-expressed genes) and generate a similar output (candidate regulators) using a non-ambiguous subset from Factorbook [46] (Materials and Methods). iRegulon identifies the correct TF at the first position in 17/30 cases while the other tools on average detect only 5. 1/30 TFs at the first position (Fig. 2D, Table S1). Interestingly, the improved performances of iRegulon are not only due to the large PWM collection and the motif2TF mapping. Indeed, iRegulon still outperforms the other methods when using only the JASPAR collection and disabling the motif2TF step (Fig. S2C) or vice versa, when manually promoting similar motifs in the other tools to the correct TF (dashed bars in Fig. 2D). As expected, the true positive target gene recovery is significantly higher when iRegulon uses a 20 kb search space around TSS compared to using only the proximal promoter (Wilcoxon rank-sum paired test, p-value = 0. 004) (Fig. S2D). We conclude that the core motif discovery framework of iRegulon is better than other tools, and that the large motif collection and the motif2TF step deliver a marked step forward in TF identification performance. In the validation and benchmark analyses above we used gene sets derived from ENCODE ChIP-Seq data as input for iRegulon. In this section, we explore more realistic types of inputs, such as co-expressed genes downstream of a TF perturbation [47]; genes involved in the same signaling pathway (e. g., KEGG [48], Reactome [49] or Gene Ontology [50]); highly connected genes in a biological network (e. g., GeneMania [51] or STRING [52]); shared targets of a common microRNA. In the first example, we applied iRegulon to a set of 171 genes that are significantly up-regulated under hypoxia [53]. iRegulon yields a top-scoring regulon that contains HIF1A as master regulator, along with 94 predicted direct target genes (Fig. S5A). The predicted HIF1A targets are likely functional targets because they overlap much more (41%) with known HIF1A targets [54] than the non predicted targets (15%). More systematically, when applied to 76 co-expressed gene sets obtained after a genetic perturbation of the TF (gene sets from MSigDB [47]), the perturbed TF is recovered in 38 cases (50%) and as the top ranked master regulator in 18 cases (24%). The lower recall to detect the correct upstream TF compared to ChIP-derived gene sets is expected because not all TF perturbation experiments successfully result in significant gene expression changes of the direct target genes. Next, we analyzed a set of 161 genes involved in the NOTCH signaling pathway and identified the top two regulons to be controlled by HEY1/HEY2/HES1 and RBPJ, two major players involved in NOTCH signaling (Fig. S5B). We also analyzed 1198 genes involved in immune response (GO: 0006955), and as expected we found the IRF and REL/NF-κB regulons, with 806 and 711 direct target genes respectively, highlighting their role as master regulators of the immune response (Fig. S5C). We also analyzed all 2233 TF-centered subnetworks within protein association networks and found enrichment of direct targets for 151 (13. 2%) and 159 TFs (14. 6%) for GeneMania and STRING networks, respectively, indicating that transcriptional interactions are partially represented in protein-protein interaction networks as well (Fig. S5D). Finally, we analyzed 159 sets of known microRNA targets, for which iRegulon identified significant cross-talks (feed-forward loops) between the predicted TF and microRNA regulons (Fig. S5E). While previous methods have thus far been validated and applied to co-expressed gene sets derived from gene expression profiling, here we show that motif discovery with iRegulon can quickly identify master regulons on diverse types of gene sets, as long as a small fraction of the input set is directly co-regulated by the same TF. We now applied iRegulon to study the gene regulatory network downstream of the p53 tumor suppressor. p53 functions mainly, if not exclusively, as a TF which regulates the expression of hundreds of genes that in turn mediate its biological activities including induction of cell-cycle arrest, senescence and apoptosis [55], [56]. Although p53 is one of the most-studied transcription factor and hundreds of target genes have already been identified [14], [55], many aspects of its downstream network remain unresolved and a more comprehensive understanding of the p53 downstream signaling network is crucial given its importance in oncogenesis. We first determined a p53-dependent gene signature in the MCF-7 human breast cancer cell line by RNA-seq upon stabilization of p53 by the non-genotoxic small molecule Nutlin-3a [57]. This treatment resulted in significant up-regulation of 801 genes and down-regulation of 790 genes. Both up- and down-regulated gene sets were subsequently analyzed with iRegulon (Fig. 3A). The top-scoring regulon in the list of up-regulated genes is confirmed as the p53 regulon, with 307 genes predicted to be direct targets (Fig. 3A and Table S2). This indicates that p53 itself is the master regulator of the downstream network and directly controls many up-regulated genes, but not all of them (at least 38%). A Gene Ontology (GO) enrichment analysis of the 307 predicted direct targets identifies p53-related processes and pathways, such as “p53 signaling pathway” (adjusted pvalue = 3. 18e-21) or “Apoptosis” (adjusted p-value = 6. 76e-07), while the set with the remaining 494 up-regulated genes show no significant GO term enrichment (data not shown). In this particular experimental setup the master regulator, namely p53, was specifically perturbed and thus known a priori. Yet, even under such circumstances there are two important advantages of using a computational regulatory analysis with iRegulon. First, the explicit finding of the p53 motif as top ranked indicates that p53 directly controls a large portion of the up-regulated genes but not all, creating two clearly distinct subsets. Second, we discover potential p53 co-factors and secondary regulons downstream of p53. Particularly, among the 801 genes that are activated downstream of p53, we found three other regulons, one operated by activator protein 1 (AP-1, heterodimer composed of JUN/FOS/FOSL1/FOSL2), another by a Forkhead TF (FOX), and another by NF-Y (Fig. 3A, Table S3A). These secondary regulons show extensive overlap with the primary p53 regulon, indicating that these TFs may be important contributors in gene regulation downstream of p53 (Fig. 3B). The AP-1 regulon, sharing 136 genes (59% of its regulon) with the p53 regulon might indicate a prevalent co-factorship between the two proteins, something that has been reported before but never on such an extended scale [58], [59]. In addition, one of the shared p53-AP1 targets is GADD45A, a gene involved in DNA damage repair, that has been shown to be a bona fide target of both p53 and AP-1 [60]. Interestingly, two subcomponents of the AP-1 complex, FOS and FOSL1, are themselves up-regulated upon p53 stabilization, and are among the predicted direct p53 targets (Table S4). These results, together with the fact that the AP-1 motif was not enriched among the down-regulated genes indicate a positive, synergistic effect of the p53 and AP-1 regulons. Nutlin-3a treatment also resulted in 790 significantly down-regulated genes. Interestingly, the analysis of this set with iRegulon does not detect the p53 motif as enriched. It does however identify E2F as master regulator with an astounding 653 (82. 7%) predicted direct targets (Table S3B). Moreover, three E2F family members, namely E2F1, E2F2, and E2F8 are all strongly and significantly down-regulated upon Nutlin-3a treatment (around 10-fold down with p-value<1. 0E-64), indicating the marked involvement of this protein family in the repressive mechanisms of p53. Similarly, iRegulon points towards NF-Y as an important second master regulator of a large number of down-regulated genes (493 genes). Both E2F and NF-Y have been reported as important players for p53-mediated down-regulation of genes [61], [62]. This may happen through p21 regulated cyclin dependent kinases, resulting in a lack of phosphorylation of NF-Y and Rb which ultimately renders both NF-Y and E2F (through Rb) inactive [63], [64]. Interestingly, the majority of NF-Y' s predicted regulon overlaps with that of E2F, with only a very small number of genes predicted as NF-Y only targets (Fig. 3B). The enriched Gene Ontology terms of these overlapping target genes are related to cell-cycle processes, an expected result since both E2F and NF-Y have been established to regulated cell cycle-related genes, often in a cooperative manner [65]–[67]. In contrast to E2F, NF-Y itself is not down-regulated as a gene by p53 activation. However, it is possible that NF-Y is regulated at the protein level rather than at the transcriptional level in response to p53 activation. All together, these findings support the notion of an indirect rather than a direct p53 repressive process largely working through the p53-p21 axis, which affects both E2F and NF-Y [63], [68]. All together, iRegulon generates marked ideas concerning p53, which are further elaborated upon in the next section. To test the predicted p53 regulon we determined the genome-wide chromatin occupancy by p53 in Nutlin-3a stimulated MCF-7 cells using high-coverage ChIP-Seq (∼30 Million uniquely mapped reads). Fig. 4A shows the raw ChIP-Seq data for the known p53 target CDKN1A, with a very strong peak overlapping the known p53 binding site in the promoter of CDKN1A [69]. To avoid arbitrary thresholds on peak calling we used lenient peak calling settings to rank all genes in the genome according to their likelihood of being a p53 target based on ChIP peaks only (see Materials and Methods). To assess whether this ranking yields true p53 targets on top, we curated 223 bona fide p53 targets from the literature and public databases (Table S5), and indeed found these targets to be significantly enriched in the top of this ranking (Fig. 4B, p-value = 1. 40E-24). Within the same ranking, the 307 predicted p53 targets by iRegulon are nearly as significantly enriched in the top as the curated targets (p-value = 2. 60E-24), while the 494 remaining up-regulated genes are not significantly correlated with the ChIP peak data (p-value = 0. 096). Importantly, this result shows that iRegulon is not only able to identify the master regulator, but is also able to correctly distinguish between direct and indirect targets from a set of co-expressed genes. Only two up-regulated genes with a high ChIP peak, namely PLK3 and DDB2, were missed by iRegulon. About 100 up-regulated genes have a small ChIP peak but have not been predicted by iRegulon as target genes. These peaks are likely false positive ChIP peaks because they do not show p53 motif enrichment when analyzed separately (Fig. S6A–C). Finally, to compare how many targets are missed by iRegulon, and how many by ChIP-Seq, we again used the set of curated targets, and found comparable numbers of false negatives, namely six for iRegulon and five for ChIP-Seq (Fig. 4C). In the previous section we had also found that gene repression downstream of p53 is indirect through E2F, which has been shown recently to be mediated by p21 and RB [63], [68]. If this is true, then the down-regulated genes should not contain p53 ChIP peaks. To test this, we plotted the recovery of the 790 down-regulated genes along the p53 ChIP-peak-based gene ranking generated above (Fig. 4B). Similar to the indirect up-regulated genes, the down-regulated genes are completely depleted of p53 ChIP peaks (p-value = 1. 0). On the other hand, the down-regulated genes are positively correlated with E2F1 ChIP-Seq data in MCF-7 from ENCODE (Fig. S6D). When combining all the small p53 ChIP-Seq peaks that are detected amongst the down-regulated genes, the p53 motif is not found by de novo motif discovery, while the ChIP peaks of direct up-regulated targets are strongly enriched for de novo p53 motifs (Fig. S6A–C). From the ChIP-Seq validation data, we conclude that iRegulon predicts the correct master regulators (p53 and E2F) and that predicted target genes of these TFs significantly overlap with ChIP-Seq derived targets. By combining iRegulon and ChIP-Seq data, we propose a set of 110 “top targets” of p53 in MCF-7 that are directly and positively regulated. When further comparing these predicted targets to recent reports of several p53 targetomes based on combining gene expression profiles with p53 ChIP-Seq data under different experimental conditions [58], [59], [68], we could confirm many common targets, but also uncovered 56 new direct p53 target genes with our analysis (Table S6). To explore the relevance of the newly identified p53 targets in other tumor types, we applied iRegulon in a meta-analysis to about twenty thousand cancer gene signatures, i. e. differentially expressed genes obtained from cancer specific experiments. We reasoned that those target genes that are recurrently predicted across cancer gene signatures, might contribute to the tumor suppressor role of p53. We used gene signatures from GeneSigDB [70], MSigDB [71] and from gene modules generated across 91 large cancer microarray data sets (see Materials and Methods and Fig. 5A). Out of 23172 signatures, p53 is found as regulator in 709 signatures. We merged the direct p53 targets across all these signatures into a network and weighted the edges according to the recurrence of this p53-target interaction across all signatures. Many previously known p53 targets and many ChIP-Seq derived targets are recovered using this analysis (GSEA NES = 3. 01, FDR<0. 001) (Fig. S7). Of the 110 predicted p53 targets in MCF-7 cells (as defined above), 44 are also predicted as p53 target in cancer gene signatures (grey area in Fig. 5B). These genes are predicted as p53 targets by iRegulon and show a significant ChIP peak and are represented in the p53 cancer-related meta-regulon. Amongst these 44 genes, 20 were previously indicated as well established p53 targets (genes in squares in Fig. 5B). When extending the analysis and including target genes recently reported in literature [58], [59], [68], it becomes clear that most overlap coincides within this metatargetome (34/44) (Table S6). Keeping in mind that many of the p53 targets reported by others were found using different cell lines, the enriched overlap within this metatargetome can be interpreted as a sign that these genes represent a core set targeted by p53 regardless of the cell type. Interestingly, when looking at targets like RAP2B, NHLH2, SLC12A4, and ALDH3A1, they could not have been identified through motif discovery in proximal promoters only, because the p53 binding sites are located either further upstream (∼1 kb for RAP2B and ∼5 kb for ALDH3A1) or in introns (NHLH2 and SLC12A4) (Fig. 5C). Next we confirmed experimentally whether these four targets are bona fide p53 transcriptional targets. They are all induced in a p53-dependent manner in various cellular model systems including normal diploid human fibroblasts (BJ cells) and various cancer cell lines (i. e. HCT116 and MCF-7) (Fig. 5D). Except ALDH3A1, they are also all significantly induced upon exposure to the DNA damaging agent doxorubicin, a well-established p53 inducer (adjusted p-value<0. 05). Their kinetic of induction both in response to Nutlin-3a and DNA damage is comparable to the one seen with known direct p53 targets such as CDKN1A further supporting a direct role for p53 in their regulation (Fig. S8). Finally, for all except one we could confirm luciferase reporter activity of the predicted p53 enhancer region (Fig. 5E). Enhancer-reporters for ALDH3A1, NHLH2 and RAP2B show a significant induction after Nutlin-3a treatment in wild type but not in a p53 knock-down (KD) cell line (p-value<0. 05). SLC12A4 does not have a significant induction in either cell-type. Note that our positive control enhancer, namely the CDKN1A promoter, is a very responsive p53 target and likely responds to low levels of p53, which could explain the induction that is still observed even under p53 KD conditions. Functionally, these validated p53 target genes have been implicated in p53-regulated processes such as the control of cell volume, growth and movement (SLC12A4 and RAP2B) and metabolism (ALDH3A1 and NHLH2). We extended our motif discovery approach to allow the discovery of significantly enriched ChIP-Seq tracks in a set of co-expressed genes. We created a database with track-based gene rankings from a collection of 1118 ChIP-Seq experiments against 246 human sequence-specific TFs across 40 cell types and apply the same “ranking-and-recovery” enrichment calculation as employed earlier (see Materials and Methods). These and other recent resources further enlarged our motif collection to 9713 distinct PWMs (“10K collection”) (Table 1). To test whether motif and track discovery can be performed simultaneously, we combined the motif-based rankings and the track-based rankings into one enrichment analysis, although each AUC score distribution is kept separate for normalization (Fig. 6A–B). Applied to the 801 p53-dependent up-regulated gene set, the combined approach still detects p53, AP-1, NFY, and FOX in the top motifs. Both for p53 and AP-1, enriched ChIP-Seq tracks are found by the track discovery, being our in-house performed p53 ChIP-Seq in MCF-7 after Nutlin-3a (ranked first of all tracks, NES = 5. 18) and the FOSL2 ChIP-Seq tracks in MCF-7 from ENCODE (NES = 3. 30) (Fig. 6C–D, Table S7). In addition, we found five more candidate TFs with a putative role in the network downstream of p53 that were not detectable using the 6K motif collection only (Fig. 3). Three of these additional candidates, namely RFX5, NR2F2, and NFI have both their ChIP-seq track and motif enriched while two more candidates, namely p300 and TCF12 only show track enrichment (Fig. 6D). To our knowledge, no interaction of these TFs with p53 has been reported in the literature. Although the targetomes of the co-factors overlap to some extent (20–42%) with p53 targets, they have a considerably large set of target genes independent of p53. Hence, with these additional TFs added downstream of p53, we can once more explain an additional fraction of the up-regulated gene set, with all the ChIP-Seq track-derived interactions together regulating 542 of the 801 genes. RFX5 is of particular interest since the gene itself is strongly up-regulated by p53 and is in fact among the core set of 801 up-regulated genes (log2FC = 1. 9 and adjusted p-value = 1. 05E-15). RFX5 is mainly known as a regulator of MHC-II genes, and indeed, among the top predicted RFX5 target genes downstream of p53 we find HLA-F, MR1, and other genes involved in antigen and interferon-related processes. Interestingly, RFX5 has recently also been shown to act as a DNA mismatch repair stimulatory factor [72], and several p53-shared RFX5 targets, such as DDB2 and BBC3, are in fact related to DNA damage response (adjusted p-value = 6. 99E-5, Wikipathway ID: WP707) (Fig. 6E). Hence, RFX5 can be considered as a new candidate co-factor to modulate certain aspects of the p53-regulated response, and may explain why MHC-II genes are up-regulated in a p53-dependent manner. This proof-of-principle of combined motif and track enrichment paves the way towards further integration of regulatory track data and enhancer prediction data to map gene regulatory networks. We have optimized and expanded motif discovery methods and used large collections of up to 10. 000 candidate motifs to facilitate translation of motif detection results into a network biology framework. By adding this network-layer on top of cis-regulatory motifs, we could generate direct insight into a biological process, rather than producing a mere list of enriched motifs from a gene set. iRegulon outperformed existing methods at detecting the correct upstream regulator. We found that using PWMs from other species than human greatly helps motif detection in human data sets. Many TFs are conserved from human to mouse, and even from human to fly or yeast, and sometimes the yeast or fly PWM is of higher quality or better captures the specificity of DNA binding. In addition, we found that using multiple PWMs for the same TF is an advantage and leads to higher performance of TF recovery compared to using non-redundant motif collections. Our motif collection also contains an important fraction of “novel” motifs for unknown TFs. These motifs are mostly derived from whole-genome computational predictions. In some cases these unknown motifs are clustered together in the output of iRegulon alongside a known motif, and can thereby lead to candidate TF predictions, while in other cases they may represent orphan motifs (with unidentified TFs). The mixture of known and unknown motifs creates a hybrid motif detection approach, combining de novo motif discovery and pattern matching approaches. Large-scale analyses of co-expressed gene sets of different origins, including co-expression, TF binding (ChIP), protein-protein association networks and microRNA targets, suggest that by exploiting the genome sequence, together with other species' genomes and collections of consensus TF binding sites, the most relevant sub-networks that underlie observed changes in gene expression or observed genetic interactions can be reconstructed. In up to 70% of the cases, the upstream regulatory factor can be identified, along with a set of direct targets. Therefore iRegulon provides an alternative approach to probe a particular biological process when gene expression data is available but the TF is not known in advance and/or ChIP-Seq is not feasible. By combining iRegulon with RNA-Seq, the resolution of gene expression profiling and gene regulatory network mapping can be increased, allowing the characterization of any cell type, cellular response, or tumor sample, up to the single cell level. Multiple regulons are often discovered from one co-regulated gene set. This is expected because in higher vertebrates gene regulation is combinatorial, where multiple TFs cooperate, either through binding in the same CRM (called heterotypic CRMs), or in separate CRMs of the same target gene [17]. In addition, the targets of a TF can be TFs themselves, and in turn activate or repress their own targets. For example, in the p53-dependent gene set iRegulon identified not only p53 as regulator, but also a previously known co-factor AP-1 and new regulators downstream of p53 such as RFX5. Interestingly, FOS and FOSL1, important members of the AP-1 complex, and RFX5, were all identified in this study as targets of p53. These regulators can explain a large proportion of the possible target genes of p53 as being indirect and regulated by another TF. When we extended our ranking-and-recovery framework to include more than one thousand ChIP-Seq data tracks, we also found the respective ChIP-Seq peaks for AP-1, RFX5, and several other co-factors as significantly enriched in the p53 downstream network. The joint finding of both a motif and a track for the same transcription factor strongly increases the confidence for these factors to play a role in the network as master regulator (i. e., directly controlling many target genes). Nevertheless, we envision that in most cases the motif enrichment alone, without any track enrichment, can directly lead to candidate master regulators, because ChIP-Seq data is condition-specific and is currently available for relatively few transcription factors. The absence of a regulator in the output of iRegulon, when neither a motif nor a track is enriched, can also be informative. For instance, neither the p53 motif nor its ChIP-Seq track are found enriched among the down-regulated genes, leading to the hypothesis that p53 does not act as a direct repressor, but only as an activator. Rather, iRegulon points to E2F as the master regulator of the down-regulated genes, both by its motif and track. This finding can be explained as indirect down-regulation of E2F targets and has recently been experimentally established: p21 controls RB1-mediated repression of E2F targets, including E2F family members themselves, thereby reinforcing this signal further [63], [68]. Our experimental findings on the p53 regulon were obtained in MCF-7 breast cancer cells. Usually, one iRegulon analysis is focused on one biological process, and predicts transcriptional targets that are relevant in that particular cell type or condition under study. We show that it is also possible to apply iRegulon more systematically on multiple signatures to identify cancer-related ‘meta-regulons’. They often represent the canonical, high-confidence target genes and agree well with ENCODE ChIP-Seq data (Fig. S7). This shows that relevant TF-target interactions can be identified purely from the genome sequence, thereby creating a valuable resource for less studied TFs. Three predefined regulatory search spaces are used in this manuscript from small to large regions: 500 bp upstream of TSS [TSS−500 bp, TSS]; 10 kb around TSS [TSS−5 kb, TSS+5 kb]; 20 kb around TSS [TSS−10 kb, TSS+10 kb]. If another gene is located within the upstream region, then the region is cut where this neighboring gene begins or ends (depending on which strand this gene is located on). Coding exons are excluded from the search space to avoid bias towards these exons through conservation. Notice that there can be multiple regions per gene (various upstream regions for alternative transcripts, and multiple introns) (see example in Fig. S1). When multiple regions are scored for a given gene, the rank of the highest ranked region is taken into account as the final rank of the gene. Motif detection relies on an offline scoring step whereby every gene in the human genome, along with orthologous sequences in ten other vertebrate genomes, is scanned with Cluster-Buster [33] for homotypic clusters of motifs using a library of N position weight matrices (PWMs), generating a database of N ranked lists of genes, each with the most likely genomic targets of a motif at the top of the ranking. As in the case of motif detection, TF ChIP-Seq track detection also relies on an offline scoring step whereby every gene in the human genome is scored with M sets of ChIP-Seq peaks (broad or narrow), generating a database of M ranked lists of genes, each with the most likely genomic targets of a TF at the top of the ranking. Our motif enrichment analysis differs from standard gene set enrichment methods such as GSEA, which uses Kolmogorov-Smirnov statistics [71]. In our method, we calculate the top enrichment of a single gene set over Nmotif genomic rankings while gene set enrichment methods assess the significance of many gene sets for one genomic ranking. Enrichment is determined by the Area Under the Recovery Curve (AUC) of the cumulative recovery curve for the input set, along the whole-genome ranking. As we are mostly interested in highly ranked genes, the AUC is computed in the top of the ranking (default set to 3%, see Fig. S2B for validation) for all PWMs or tracks of the collection. A Normalized Enrichment Score (NES) for a given motif/track is computed as the AUC value of the motif/track minus the mean of all AUCs for all motifs (or tracks), and divided by the standard deviation of all AUCs. When the distribution of AUCs follows a normal distribution then the NES score is a z-score indicative of the significance. The default NES cutoff in iRegulon is 3. 0, corresponding to FDR between 3% and 9% (Fig. S3). For each enriched motif, the candidate targets are selected as the optimal subset of highly ranked genes compared to the genomic background and to the entire motif collection as background. This step is illustrated in Fig. 1B. The target gene recovery is plotted along the whole-genome ranking for a given motif (blue curve) and compared to the average recovery + (2× standard deviation) (red curve) for all motifs in the collection. Similarly to the GSEA approach [71], the leading edge corresponds to the rank where the difference between the signal (blue curve) and the background (red curve) is maximal within the top ranked genes (the latter is defined by the Rank Threshold parameter). The input genes that have a better ranking than the rank at the leading edge are predicted as target genes for the given motif or track. Enriched motifs are linked to candidate TFs, which could potentially bind to the motif. If we use only the direct annotations, only a small fraction of motifs (20%) can be associated to human TFs (521 TFs with “6K” collection, 944 TFs with “10K” collection). We developed a database, which we term the motif2TF database, corresponding to a network of associations between motifs and TFs where motif-TF edges correspond to all motif-TF direct annotations (from different species), TF-TF edges are defined by homology (using Ensembl Gene Trees [43], [44]), and motif-motif edges are defined by motif similarity, defined by the Tomtom p-value [45]. For each motif all possible TFs are associated following different paths in the motif2TF network. In the plugin at the client side, these TFs are ranked, prioritizing directly annotated TFs, then the TF present in the input set, then the ones that are found by gene homology and finally the TFs found using motif similarity. Figure 1C illustrates the different possible paths on a motif2TF subnetwork. Motif M1 is not directly annotated to any TF (so it can be part of the unknown motif collections), but is similar to two other motifs, namely M3 and M4, both of which are directly annotated. Motif M4 is directly annotated to a human TF (TF1), while M3 is a motif annotated for a TF from another species (TF7). Three TFs in human (TF1, TF8, TF6) are possible orthologs of TF7. In this example, the link between M1 and TF1 would go via the path through M4, which is the shortest and best path (rather than via M3 and TF7). For M1, motif2TF returns TF1, TF6, and TF8 as candidate TFs, which are subsequently ranked. The second example is for motif M2 which is annotated for TF5 in another species. Three human transcription factors (TF2, TF3, TF4) are possible orthologs of TF5, which may represent for example a family of homologous TFs such as GATA factors, E2F factors, or ETS factors. In such a TF family, the consensus motif may indeed be shared by multiple family members and therefore iRegulon may return multiple or all family members as candidates. When multiple TFs are returned, we give priority to a TF when it is part of the input genes. In this example, TF2 will be preferentially associated to M2 as it belongs to the input genes (encoded by TG5 in the Figure). ChIP-Seq data was downloaded as hg19 aligned bed files (view = peaks) from the TFBS ENCODE collections available from the following servers: http: //hgdownload. cse. ucsc. edu/goldenPath/hg19/encodeDCC/wgEncodeSydhTfbs/ http: //hgdownload. cse. ucsc. edu/goldenPath/hg19/encodeDCC/wgEncodeHaibTfbs/ http: //hgdownload. cse. ucsc. edu/goldenPath/hg19/encodeDCC/wgEncodeUchicagoTfbs/. Almost one thousand files (999) were downloaded corresponding to 160 sequence-specific TFs (TFSS): 672 files for HAIB (Hudson Alpha Institute), 323 for SYDH (Stanford/Yale/USC/Harvard) and 6 files for Uchicago. Files corresponding to Input and RNA Polymerase 2 (“Pol2”/“Pol2 (phosphoS2) ”) were not downloaded. 115 TFs are detectable in iRegulon (i. e., at least one motif in the collection of 6383 motifs can be connected to the TF), corresponding to 786 ENCODE datasets. Each query set consists of the top 200 target genes presenting a ChIP peak in a predefined search space, i. e., for each search space tested (500 bp upstream of TSS; 10 kb around TSS; 20 kb around TSS), we define a different set of target genes, so that each target gene contains a ChIP peak within the chosen motif search space. The ChIP-Seq scoring of the genes has been done as mentioned earlier in the Track-based rankings section. Finally, note that our motif collection does not contain PWMs derived from these datasets (so we rely on other, previously curated PWMs to identify the correct TF). The Factorbook dataset collection is a subset of this ENCODE selection corresponding to 254 ChIP-Seq files (121 from HAIB, 129 from SYDH and 5 from Uchicago), inferred from the list of signatures published in the Table S1 of the FactorBook reference publication [46]. 126 out of these 254 FactorBook signatures have the canonical motif corresponding to the ChIP' ped TF. From these we randomly selected one signature per TF for which the canonical motif was predicted as top 1 by their motif discovery pipeline (inferred from Table S1A [46]). The list of the 30 used datasets is presented in Table S3. Different types of control gene sets were selected, namely: from ENCODE ChIP-Seq we used (1) genes without a ChIP-Seq peak in the corresponding search space; (2) TF neighborhoods for 1150 TFs, containing for each TF all the genes within 5 Mb flanking the TF; and (3) 1161 random signatures. Datasets are available on our laboratory website (http: //www. aertslab. org). We also got similar performances using 631 uniformly reprocessed ChIP-Seq data generated in NarrowPeak format by the ENCODE Analysis Working Group downloaded from http: //genome. ucsc. edu/cgi-bin/hgFileUi? db=hg19&g=wgEncodeAwgTfbsUniform (data not shown). The classical motif discovery algorithms that originated in the late 1990s can be put in two categories: string-based or enumeration methods and matrix-based approaches. The string-based approaches rely on the detection of statistically over-represented words (oligonucleotides or spaced motifs) compared to a given background [83]–[88]. Matrix-based approaches make use of position weight matrices (PWMs) as a predictive model for TF binding sites, which can be graphically represented as a motif logo [89], and use optimization algorithms (Expectation-Maximization [90], greedy algorithm [91], [92] or Gibbs sampling [93]–[95]) to find the most common motifs to all input sequences. Most of these methods performed well on yeast or bacterial promoter sequences, but they showed limited performances when applied to mouse or human [96]. These methods could be improved by phylogenetic footprinting [97]–[103] and by applying genome-scale methods that exploit the entire gene expression data set rather than a set of co-expressed genes [104]–[106]. Current developments have on the one hand focused on the application of the early algorithms to ChIP-Seq data [107]–[111], and on the other hand on the application of motif discovery to gene sets, with the aim to increase the performance in higher eukaryotes such as fly, mouse and human, using large sequence search spaces. This category of PWM enrichment methods is represented by phylCRM/Lever [30], DIRE [80], [112], PASTAA [32], [113], PSCAN [114], Allegro [115], HOMER [116], OPOSSUM [117] and i-cisTarget [28]. They all use libraries of candidate PWMs and apply PWM enrichment statistics, often combined with other cues, such as comparative genomics and TF binding site clustering. By using libraries of PWMs for known TFs (e. g., PWMs derived from protein binding microarrays), these methods promote a TF to a candidate master regulator of the gene set when its PWM is found enriched. We used all methods in this category of PWM enrichment methods that are available online, that can work on human gene sets, and that can be practically performed on 30 sets of 200 human genes. Thirty gene sets from FactorBook were selected for motif discovery tool comparison (Fig. 2D, Table S1). These gene sets have been selected because the motif of the ChIP' ped TF was detected as top enriched motif in the top 500 peaks in FactorBook. We extracted the top 200 genes having the highest peaks in their 20 kb region around the TSS. The comparison was performed on TF and motif recovery using the parameters indicated in Table S3. The parameters were left to default and when possible, we only adjusted the parameters to allow for larger upstream regions (when possible we choose TSS+−10 kb). iRegulon was compared to eight other publicly available motif enrichment tools, namely OPOSSUM [117], DIRE [80], [112], PASTAA [32], [113], PSCAN [114], Clover [16], AME [118], Allegro [115] and HOMER2 [116] (in the case of Homer2, de novo and known motif discovery are performed simultaneously but we consider them as different approaches and validate them separately). We selected these tools because they mostly take as input a set of human co-expressed genes, and they all return, at least to some extent, information on which TF could be regulating the input genes. For this reason, it not feasible to compare iRegulon with classical de novo motif discovery methods (e. g., MEME-like methods) because such methods are intractable on large human gene sets (e. g., 200 genes×20 kb×10 species represents a sequence set of 40 Mb), and they result in new motifs rather than candidate TFs. We also attempted to use SMART [119] but we did not succeed in running the software. For tools that require regulatory sequences as input (AME and Clover) we used the same sequences as used by iRegulon. For some tools like Clover, it is theoretically possible to use a large search space but one run on one dataset takes too long (∼17 hours), and therefore we limited the analysis to 500 bp promoter sequences. In the case of AME, we found no positive results with a large search space (data not shown), so we show the results with the default search space. For comparison, we used the number of motifs/TFs found in top 1 and within top 5 positions. The total number of detected motifs was not reported for comparison, because some tools use more stringent thresholds than others. All these tools rely on the available motif annotation to identify the candidate TF such as Jaspar (J) or Transfac (T). However, we also manually re-associated the detected motifs to candidate TFs (mainly by comparison of the detected motif with the FactorBook motif) (see column “USING SIMILARITY” in the Table S3). For Homer2,14 motifs that are derived from ENCODE ChIP-Seq data matching the actual Factorbook ChIP-Seq data were discarded from their in-house PWM collection to avoid over-fitting (indeed, iRegulon does not include FactorBook PWMs either, nor do any of the other tools). Note that for the other large-scale analysis (e. g. full ENCODE analysis), we use a command-line version of iRegulon. At the client side, iRegulon is implemented in JAVA as a Cytoscape plugin, which can be downloaded from http: //iregulon. aertslab. org. The iRegulon plugin is connected to the server-side daemon over the Internet. The iRegulon server-side daemon is implemented in Python and uses MySQL to store and query the PWM-based whole-genome rankings (see below). After submitting a gene set or network to the service, the results are returned to the client, and this happens on-the-fly, and takes about one minute. The user can browse through the motif discovery results, select a TF among the prioritized list of TFs, and add upstream regulators and direct regulator-target ‘edges’ to the input gene set or network under study. A detailed description on the use of the plugin is provided in Figure S4. In addition, the plugin allows querying cisTargetDB to obtain the meta-regulon for a given TF, i. e. targets found recurrently predicted for this TF by iRegulon across thousands of signatures/gene sets. iRegulon results were obtained by running the Cytoscape plugin v0. 97 on Cytoscape 2. 8. 1. The current version of iRegulon (1. 2) supports the “10K” motif collection and the track discovery. The source code of the iRegulon plugin is also available from the iRegulon website (http: //iregulon. aertslab. org). iRegulon was applied in batch (i. e., using the GMT file format as input for the command line version of iRegulon) to 3447 signatures in GeneSigDB (version 4), 6753 signatures from MSigDB (version v3 collection 2) and 12972 bi-clusters we obtained in-house. Bi-clustering was performed with Ganesh clustering algorithm [120], [121] using default settings to 91 microarray datasets. The 91 datasets were retrieved as normalized (fRMA) microarray data from InsilicoDB [122]. iRegulon results on all these gene sets is stored in a MySQL database, from which all summaries per motif and subsequently per TF are computed, resulting in a meta-regulon per TF. In a meta-regulon, each target gene is annotated with a number that represents the number of gene sets where the TF is found enriched and the gene is among the optimal subset of direct targets. GO enrichment analysis was performed using DAVID [123], [124] or BINGO [125]. GSEA analysis on ChIP-Seq data was performed to avoid arbitrary peak score cutoffs. The genome was ranked according to the MACS ChIP-peak score (score range between 0 and 1517. 33 for p53) within an area of 20 kb around the TSS of 22284 RefSeq genes. Functional categories found enriched for co-factors of p53 were calculated by DAVID and WebGestalt [126] based on Gene Ontology and KEGG pathways. Cells were kept in culture at 37°C, with 5% CO2 and in RPMI medium (+ L-glutamate, Gibco) supplemented with 10% fetal bovine serum (Invitrogen), 0. 4 mM sodium pyruvate (Gibco), 100 µm/ml penicillin/streptomycin (Invitrogen), 1× non-essential aminoacids (Gibco) and 10 µg/ml Insulin (Sigma). p53-Wild-Type MCF-7 cells were plated onto 24-well plates (60000 cells/well). The next day, cells were either stimulated with 5 µM Nutlin-3a or left untreated. After 24 h, cells were washed in PBS (Gibco) and prepared for RNA extraction according to the RNeasy protocol (Qiagen), yielding around 2 µg of total RNA per sample. The quality of the RNA samples were checked using a Bioanalyzer 1000 DNA chip (Agilent) after which libraries were constructed according to the Illumina TruSeqTM RNA Sample preparation guide. Final libraries were pooled and sequenced on the HISeq 2000 (Illumina), generating approximately 30 million reads of 50 bp length. After removing adapter sequences reads were mapped to the human reference genome (hg19) using TopHat v1. 3. 3 [127] with default settings. Reads were aggregated with HT-Seq (–str = no parameter, version 0. 5. 3p3) using the human RefSeq annotation, release 42. DESeq [128] was used to normalize and to calculate differential expression between Nutlin-3a stimulated and non-stimulated samples. A final list of differentially expressed genes was obtained using adjusted p-value<0. 05 and |log2FC|>1. The threshold of 2-fold up-regulation was supported by the observation that the strongest enrichment of the targets from the KEGG p53 signaling pathway is observed among the top 648 up-regulated genes (GSEA leading edge corresponds to log2FC = 1. 182). p53 wild-type MCF-7 cells were seeded at a density of 5 million cells per 15 cm dish and grown ON at 37°C to 80–90% confluency. Cells were then stimulated with 5 µM Nutlin-3a for 24 h. ChIP samples were prepared following the Magna ChIP-SeqTM preparation kit using the p53 antibody (DO-1, SCBT). Per sample, 5–10 ng of precipitated DNA was used to perform library preparation according to the Illumina TruSeqTM DNA Sample preparation guide. In brief, the immunoprecipitated DNA was end-repaired, A-tailed, and ligated to diluted sequencing adapters (dilution of 1/100). After PCR amplification with 15–18 cycles and gel size selection of 200–300 bp fragments, the libraries were sequenced using the HiSeq 2000 (Illumina). Cleaned reads were mapped to the human reference genome hg19 (UCSC) using bowtie (v2. 0. 0-beta3) with the addition of parameter –local, allowing for further soft clipping of the reads. Reads with a mapping quality below 4 were removed. Peak calling was performed using MACS (version 1. 4. 2) [129] either with the default p-value threshold (3634 peaks) of 1. 0E-5 or using p-value<0. 05 (lenient setting to generate the whole-genome ranking). MCF-7, HCT116 (human colon carcinoma cell line) and BJ cells were treated continuously with 10 µM Nutlin-3a or a pulse of 5 µM Doxorubicin and total RNA was harvested at different time points. Reverse transcription was performed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Real Time quantitative PCR reactions were run on LightCycler480 (Roche) in 384-well format, using SYBR-Green Fast Universal PCR Master Mix (Applied Biosystems). Multiple primer pairs were tested for each target, and melting curve analysis confirmed amplification of a single product. Normalization was done with the most stable reference genes, assessed by GeNorm analysis [130]. The normalized relative fold changes were log-transformed before performing two-sided t-test to determine significance levels. The p-values were further corrected for multiple testing by very stringent Bonferroni correction. RT-qPCR primer sequences: NHLH2-fw-CACTGTGGGAGGATCTGAGC; NHLH2-rev-ATAAAGGGGCACTTCGCCTG; ALDH3A1-fw-CTGCAGGGAACTCAGTGGTC; ALDH3A1-rev-GGTACAGATCCTTGTCCAGGT; SLC12A4-fw-GGGAACAACATTCGCAGCAG; SLC12A4-rev-AGTGGCATTCGACGTGTCAT; RAP2B-fw-GCGCACAAAAGCCAAACGC; RAP2B-rev-AGACACCCTGGCCAATGCAA. MCF-7 cells (WT or p53-KD) were seeded in a 24 well plate at a density of 50 000 cells per well. After 24 h, cells were transfected using Fugene HD (Promega) in a 1∶3 ratio. 400 ng of luciferase reporter plasmid containing one of the enhancers of interest (CDKN1A, RAP2B, ALDH3A1, SLC12A4 and NHLH2) was mixed with a β-gal plasmid in a 1∶10 ratio to correct for transfection efficiency. The next day, cells were stimulated with 5 µM Nutlin-3a. After 24 h, the transfected cells were harvested and luciferase and β-galactosidase activities were measured following the manufacturer' s instructions (Applied Biosystems). The p-values were calculated using a t-test. The RNA-Seq and ChIP-Seq data are available from the NCBI GEO database (GSE47043). | Title: iRegulon: From a Gene List to a Gene Regulatory Network Using Large Motif and Track Collections Summary: Gene regulatory networks control developmental, homeostatic, and disease processes by governing precise levels and spatio-temporal patterns of gene expression. Determining their topology can provide mechanistic insight into these processes. Gene regulatory networks consist of interactions between transcription factors and their direct target genes. Each regulatory interaction represents the binding of the transcription factor to a specific DNA binding site near its target gene. Here we present a computational method, called iRegulon, to identify master regulators and direct target genes in a human gene signature, i. e. a set of co-expressed genes. iRegulon relies on the analysis of the regulatory sequences around each gene in the gene set to detect enriched TF motifs or ChIP-seq peaks, using databases of nearly 10. 000 TF motifs and 1000 ChIP-seq data sets or "tracks". Next, it associates enriched motifs and tracks with candidate transcription factors and determines the optimal subset of direct target genes. We validate iRegulon on ENCODE data, and use it in combination with RNA-seq and ChIP-seq data to map a p53 downstream network with new predicted co-factors and targets. iRegulon is available as a Cytoscape plugin, supporting human, mouse, and Drosophila genes, and provides access to hundreds of cancer-related TF-target subnetworks or "regulons". | 15,189 | 331 | lay_plos | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application is a continuation application of U.S. patent application Ser. No. 13/846,695, entitled COMBINED ACTIVE AND PASSIVE LEG PROSTHESIS SYSTEM AND A METHOD FOR PERFORMING A MOVEMENT WITH SUCH A SYSTEM, and filed Mar. 18, 2013, which is a continuation application of U.S. patent application Ser. No. 11/887,374, entitled COMBINED ACTIVE AND PASSIVE LEG PROSTHESIS SYSTEM AND A METHOD FOR PERFORMING A MOVEMENT WITH SUCH A SYSTEM, and filed Jul. 30, 2009, which is a national stage entry of International Patent Application No. PCT/SE2006/000445, entitled COMBINED ACTIVE AND PASSIVE LEG PROSTHESIS SYSTEM AND A METHOD FOR PERFORMING A MOVEMENT WITH SUCH A SYSTEM, and filed Apr. 18, 2006, the entireties of which are incorporated by reference herein and should be considered a part of this specification. BACKGROUND [0002] 1. Field [0003] The present invention relates generally to a method for a combined active and passive leg prosthesis system and a combined active and passive leg prosthesis system for replacing a missing lower extremity of an individual to perform a gait cycle (take a step) by the leg prosthesis system. The leg prosthesis system has at least one movable joint and a drive unit to drive the movable joint. [0004] More specifically, the present invention concerns a leg prosthesis system intended for a person who has a leg amputated above or below the knee joint. Thus the system may comprise an articulated ankle joint or both an articulated knee joint and an articulated ankle joint. [0005] 2. Description of the Related Art [0006] A leg prosthesis system of the type stated above can be adjusted and used by wearers of a prosthesis at different amputation levels. [0007] A person uses his entire body to perform a movement. A gait cycle, that is two steps, is divided into a support phase and a swing phase. The support phase is when the foot of the leg is in contact with the ground, and during the swing phase the foot swings freely in the air. In walking, one leg swings forward with a flexion in hip, knee and ankle joint while at the same time the arm on the opposite side swings forward. The arm on the same side swings backward and helps to increase the forward force and promotes balance. When the foot of the leg touches the ground, heel touching ground, the weight of the body is supported and the forward fall is braked. The leg supports the body and drives it forward until the next leg takes over in placing the heel on the ground. [0008] If one or more joints and muscles are missing, in this case in lower extremities, there will be imbalance and more energy will be consumed. The more joints that are missing and the higher the amputation level, the more energy is required to perform a gait cycle. [0009] A gait cycle, in this case for a leg prosthesis system, can be identified as the movement performed by leg prosthesis system from placing the heel on the ground to the next placing of the heel on the ground with the same leg. [0010] A leg prosthesis system according to the invention can be used to facilitate different types of gait cycles for the prosthesis wearer, for instance to walk at varying speeds, to climb or descend stairs or walk on inclined surfaces. [0011] With the leg prostheses that are available on the market, it is difficult or even impossible for a leg prosthesis wearer to perform certain types of gait cycles. [0012] The leg prosthesis systems that are being sold today are completely passive, that is no external energy is supplied to the prosthesis in addition to the prosthesis wearer's kinetic energy. A passive prosthesis can only lock, dampen and decelerate a movement. With a good passive leg prosthesis system according to prior art technique, a prosthesis wearer can handle most everyday situations, such as sit down, stand, walk on flat ground and descend stairs and slopes. [0013] However, the energy that can be supplied to a passive leg prosthesis system by the prosthesis wearer is in most cases not sufficient to perform more energy-consuming gait cycles, such as stand up from a sitting position, quick increase of speed or climb a staircase or a steep slope. Another problem is to ensure ground clearance to reduce the risk of stumbling. [0014] The manufacture and development of active leg prosthesis systems has only started recently, that is prostheses comprising motors and power sources. By supply of power, the leg prosthesis system helps the prosthesis wearer to perform certain gait cycles such as climb a staircase. [0015] The technique chosen to supply power in active leg prosthesis systems, according to prior art technique, has resulted in problems, such as high movement of inertia in moving the movable joints of the leg prosthesis system. All parts have to be driven when performing a gait cycle and the energy consumption in the leg prosthesis system will be high. [0016] There is thus a need to improve prior art and many of the leg prostheses that are currently available on the market. SUMMARY [0017] An object of the present invention is to provide a leg prosthesis system which eliminates one of the problems described above in a simple and effective manner. [0018] Another object of the invention is to provide a method of performing gait cycles with improved performance by means of a leg prosthesis. [0019] The above objects and other objects that will be evident from the following description are achieved by a device and a method according to the claims. [0020] A leg prosthesis system according to the invention comprises at least one movable joint which can be switched between being actively driven or being passively braked. Furthermore the leg prosthesis system comprises a control system for controlling the active and the passive unit in the movable joint. A number of transducers and sensors arranged on the leg prosthesis system supply input data to the control system, and a power source supplies power to control system, motors and transducers. [0021] A leg prosthesis system according to the invention comprising control system, power source and transducers can be provided with an ankle joint comprising an active and a passive unit and/or a knee joint comprising an active and a passive unit. [0022] The leg prosthesis system according to the invention can also be adjusted to a prosthesis wearer who lacks both lower extremities, that is who is double-leg-amputated. Each leg prosthesis system may, but need not, comprise one common or two separate or communicating control systems. [0023] According to one aspect of the present invention, a combined active and passive leg prosthesis system is provided to replace a missing lower extremity of an individual to perform a gait cycle by means of the leg prosthesis system. The leg prosthesis system comprises at least one movable joint and an active drive unit to supply power and drive the movable joint. Moreover the active drive unit is disconnectable from driving relation with the movable joint. By the active drive unit being disconnectable, low moment of inertia can be achieved in the movable joint. The forward force in the natural swinging motion created by the wearer's body is then sufficient to move the joint. When the joint is disconnected from driving, it is possible to brake the movement of the joint when required. Braking of the movable joint performed by a passive brake unit may be varied in braking force and be varied from a completely unbraked (freely swinging) to a fully braked (locked) joint. The interaction between active driving and passive braking of a joint gives a prosthesis wearer a good possibility of performing gait cycles with improved performance. An active drive unit which can be run in two directions makes it possible to perform a movement in both directions of the joint if, in addition, it is possible to disconnect the active drive so as to perform a free movement of the joint which can be controlled by the passive brake unit. Active and passive movements are a natural part of the gait pattern, which means that a system having such possibilities can more easily imitate the energy-saving way of the human body to perform movements. [0024] Preferably, the active drive unit of the leg prosthesis system is disconnected from driving relation with the movable joint during part of the gait cycle. For instance, the active drive unit can be disconnected during both the swing and the support phase. [0025] Preferably, a passive brake unit acts on the movement of the joint when the active drive unit is disconnected from the joint. By using the passive brake unit when the active drive unit is disconnected, the energy-saving way of the human body to perform movements can be imitated. [0026] Preferably, the leg prosthesis system according to the invention also comprises a control system adapted to control the active drive unit of the movable joint. The control system makes it possible to control direction, force and speed of the active drive unit. The control system also makes it possible to activate driving only at points of time when the leg prosthesis system needs supply of extra power. [0027] Preferably, the control system according to the invention is also adapted to control the passive brake unit of the movable joint. A control system can be used to adjust the brake force from a completely unbraked (freely swinging) to a fully braked (locked) joint. If the control system controls both the active drive unit and the passive brake unit, a number of advantages can be achieved since the leg prosthesis system according to the invention can use the properties of both the active drive unit and the passive brake unit and also the possibility of switching between driving and braking the movable joint. [0028] Preferably the leg prosthesis system comprises both a movable knee joint and a movable ankle joint. A prosthesis wearer who lacks a knee joint, that is who is amputated above the knee joint, needs a leg prosthesis which has both knee joint and ankle joint. A prosthesis wearer who lacks both lower extremities, that is who is double-leg-amputated, needs a leg prosthesis system with at least two movable joints. [0029] Preferably, the knee joint and the ankle joint are each arranged with a disconnectable active drive unit and a passive brake unit as well as a common control system to control the movements of knee joint and ankle joint in a synchronized manner. In double amputation, two separate or communicating control systems may, but need not, be used. A leg prosthesis system which is made up in this manner can use active and passive techniques for both knee joint and ankle joint. When besides a control system coordinates the movement of the knee joint and the ankle joint, considerable improvements can be achieved compared with a completely passive prosthesis, but also compared with an active prosthesis which comprises a knee joint or a knee joint and an ankle joint which are active but not coordinated with a common control system. [0030] Preferably the leg prosthesis system according to the invention comprises transducers for input data to the control system. Input data can be sent to the system from, for instance, a key set, transducers, sensors (speed, position, angle, pressure), nerve sensors etc. [0031] Preferably, the knee joint and the ankle joint move by switching between the associated active drive unit and passive brake unit during a gait cycle. [0032] A leg prosthesis system according to the invention with a movable knee joint and ankle joint which controls the direction, force and speed of the current control system for the active drive unit and the brake force for the passive brake unit in each joint independently of the other joint thus ensures an optimized and coordinated movement of the leg prosthesis system. [0033] The control system may use information from the entire leg prosthesis system, for instance information from the knee joint when the ankle joint is to be controlled and vice versa. [0034] According to one aspect of the present invention, a method is provided for performing a gait cycle with a combined active and passive leg prosthesis system, which replaces a lacking lower extremity of an individual. The leg prosthesis system comprises at least one movable joint and an active drive unit which supplies power and drives the movable joint. Moreover, the active drive unit is disconnected from driving relation with the movable joint during part of the gait cycle. By disconnecting the drive unit, it is, for instance, possible to use the natural swinging movement during the swing phase in walking. Thus the drive unit need not be adjusted to accomplish movements equivalent to free swinging. The active drive unit can instead be optimized for driving, and energy saving can be ensured since the drive unit is not activated during the entire time of use of the prosthesis. [0035] Preferably the method comprises braking the movement of the joint, with a passive brake unit of the leg prosthesis system according to the invention, when the active drive unit is disconnected from the joint. When the active drive unit is disconnected and the passive brake unit is connected, the prosthesis can move freely by the forward force in the joint in question created by the wearer's body, or the movement of the joint can be braked. [0036] Preferably the method comprises controlling of the drive unit and/or the brake unit of the movable joint by a control system of the leg prosthesis system according to the invention. By using a control system, it is possible to disconnect driving and/or activate the passive brake unit at the correct point of time or taking the outer circumstances into consideration. The braking in the leg prosthesis system is a property which advantageously is coordinated with the driving of the system to achieve a good result. [0037] Preferably the method comprises supplying input data to the control system from transducers of the leg prosthesis system. Input data is sent, for instance, from a key set, transducers, sensors (which record speed, position, angle, pressure), nerve sensors etc. [0038] The method preferably comprises controlling in a synchronized manner the movements of a knee joint and an ankle joint of the leg prosthesis system. The knee joint and the ankle joint each have a disconnectable active drive unit and a passive brake unit as well as a common control system. In a leg prosthesis with both knee joint and ankle joint, improved possibilities are achieved for the prosthesis wearer since a control system which is common to both joints can coordinate the movements of knee joint and ankle joint. [0039] The knee joint and the ankle joint preferably move by switching between the associated drive unit and brake unit during a gait cycle. [0040] Preferably the method comprises controlling, separately or in a communicating manner, the movements of at least two movable joints of leg prosthesis systems adjusted to a double-leg-amputated prosthesis wearer. [0041] The control system may use information from the entire leg prosthesis system, for instance information from the knee joint when the ankle joint is to be controlled and vice versa. [0042] The method preferably comprises driving and braking of knee joint and ankle joint, respectively, during a gait cycle. BRIEF DESCRIPTION OF THE DRAWINGS [0043] The invention will now be described in more detail with reference to the accompanying drawings, in which [0044] FIG. 1 is a perspective view of a leg prosthesis system according to the invention, [0045] FIG. 2 illustrates the support phase of a gait cycle on a flat surface, [0046] FIG. 3 illustrates the swing phase of a gait cycle on a flat surface, [0047] FIG. 4 illustrates the support phase of a gait cycle when climbing a staircase, dashed lines indicating a corresponding gait cycle with a passive/rigid foot, [0048] FIG. 5 illustrates the swing phase of a gait cycle when climbing a staircase, [0049] FIG. 6 illustrates the support phase and the swing phase of a gait cycle when descending a staircase, [0050] FIG. 7 illustrates the support phase of a gait cycle when climbing a slope, [0051] FIG. 8 illustrates the swing phase of a gait cycle when climbing a slope, [0052] FIG. 9 illustrates the support phase of a gait cycle when descending a slope, [0053] FIG. 10 is a side view in cross-section of a knee in an active straightening position, [0054] FIG. 11 is a side view in cross-section of a knee in a passive braking position, [0055] FIG. 12 is a side view in cross-section of a foot in an active position according to the invention. DETAILED DESCRIPTION [0056] FIG. 1 shows a leg prosthesis system 1 with both knee joint 2 and ankle joint 3 according to the invention and a method of performing a gait cycle with a leg prosthesis system. A prosthesis wearer can attach the prosthesis to the amputated leg by means of the leg-enclosing socket 13. Furthermore the socket 13 is attached to the movable knee joint 2 in a suitable manner and the knee joint is connected to the ankle joint 3 by interconnecting elements 12 or the like. A foot prosthesis 14 is attached to the ankle joint 3 and can turn about the ankle joint 3. Additional components that may be included in a leg prosthesis system are shock absorbers, angularly adjustable couplings etc. [0057] Most of the people with an amputated leg have lost their leg below the knee joint. The present leg prosthesis system and/or method can be used by prosthesis wearers who need a prosthesis with both knee joint and ankle joint, but the invention can also be used for a prosthesis with only an ankle joint or only a knee joint. The leg prosthesis system and/or the method can also be used by prosthesis wearers who lack both lower extremities, that is who are double-leg-amputated and need a leg prosthesis system with at least two movable joints. One common or two separate and/or communicating control means may be used. [0058] FIG. 2 shows the support phase for a gait cycle on a flat surface. When placing the heel on the surface, FIG. 2.1, the body weight of the prosthesis wearer is applied to the leg prosthesis system. The knee joint then allows flexion and the foot is plantar flexed, FIG. 2.2, that is the foot blade moves away from the lower leg. Body weight and muscular strength help to straighten knee joint and ankle joint to centered standing, FIG. 2.3. In FIG. 2.4, the foot blade is compressed and energy is returned in FIG. 2.5. When performing this movement, from FIG. 2.1 to FIG. 2.5, the leg prosthesis system is completely passive, passive braking of both ankle joint and knee joint. The joints are rotated by means of body weight and muscular strength from the remaining lower extremity. For extra power in the gait, for instance when walking faster, the active drive unit in the foot can be used in the position in FIG. 2.6 to push away. [0059] FIG. 3 shows the swing phase in a gait cycle. FIG. 3.1 corresponds to FIG. 2.6 and when initiating a swing phase, FIG. 3.3, the active part of the ankle joint performs a dorsal flexion, that is the foot blade moves towards the lower leg. This dorsal flexion occurs to give the prosthesis wearer ground clearance, a safe distance between the foot and the ground to prevent stumbling. A passive foot does not manage the dorsal flexion from FIG. 3.2 to FIG. 3.3 but this movement of the foot blade requires some kind of drive. The knee joint performs the swinging movement by using the forward force created by the wearer's body, and the passive braking controls the movement. When performing this movement, from FIG. 3.3 after the dorsal flexion to FIG. 3.5, the leg prosthesis system is completely passive, passive braking of both ankle joint and knee joint. To provide extra force to the step, for instance when walking faster, the active drive unit in the knee joint can be used in the position in FIG. 3.4 to straighten the knee joint and move the lower leg forward more quickly. [0060] To climb a staircase or slope, as illustrated in FIG. 4, FIG. 5, FIG. 7 and FIG. 8, it is important for the knee joint and the ankle joint to cooperate. More energy is required in climbing, which means that cooperating active drive of both ankle joint and knee joint can advantageously help to perform the movement. [0061] When descending a staircase or slope, as illustrated in FIG. 6 and FIG. 9, the passive braking in both knee joint and ankle joint cooperates. [0062] FIG. 4 to FIG. 6 illustrate the climbing of a staircase. FIG. 4 shows the support phase when climbing a staircase. In FIG. 4.1 the foot is positioned on the step, and balance is achieved. The leg prosthesis system and/or the use of the method according to the invention then push the prosthesis wearer upwards, FIG. 4.2, to centered standing, FIG. 4.3. The control system makes it possible for the knee joint and the ankle joint to cooperate. The active drive unit in the knee joint strives to straighten the knee joint while at the same time the active drive unit in the ankle joint presses the front of the foot towards the ground, FIG. 4.2. In this manner, the ankle joint helps to straighten the knee joint, thereby reducing the energy consumption. [0063] FIG. 4 illustrates the consequences of a passive foot, dashed lines, in combination with an active knee joint. The passive foot gives a higher knee joint position and the gait will be higher than it need be, and it will be more difficult and require more energy for the user to raise himself up on the step. The contact point of the passive foot on the step is moved forwards compared with a foot which can perform a dorsal flexion, which results in also the center of gravity of the body having to be moved forwards. The solid lines indicate how climbing a staircase can be performed using a leg prosthesis according to the invention. [0064] FIG. 5 illustrates the swing phase when climbing a staircase. Also in this case the active drive units in the knee joint and the ankle joint are used. To prevent the prosthesis wearer from hitting the step with his foot and stumbling in the swing phase when climbing a staircase, it is important that the knee joint and ankle joint create a safe distance to the staircase. This is done by the active drive of the knee joint bending the joint and the drive of the ankle joint performing a dorsal flexion of the foot, FIG. 5.1. The leg prosthesis system according to the invention has then created a safe distance to the staircase and also a good starting position for positioning for the next gait. FIG. 5 shows the consequences of a completely passive system, dashed lines. The knee joint does not bend the foot away, and the foot instead bumps into the staircase. [0065] FIG. 6 illustrates the descending of a staircase. Here both knee joint and ankle joint are mainly passive. The movement, FIG. 6.1 to FIG. 6.3, brakes the fall of the body by means of the passive brake units in knee joint and ankle joint. The dashed lines indicate the consequences of a passive foot which is not capable of performing a dorsal flexion. The active drive units can optionally be used to help straighten knee joint and ankle joint in the swing phase. [0066] FIG. 7 to FIG. 9 illustrate walking on a very sloping surface. The leg prosthesis system according to the invention then functions in the same way as when climbing a staircase. The angle of knee joint and ankle joint is the only thing that distinguishes the climbing of a staircase from walking on a very sloping surface. When walking on a slightly sloping surface, the walking can be more resembled to walking on flat ground. [0067] FIG. 4, FIG. 5, FIG. 7 and FIG. 8 illustrate ordinary situations which require much energy to be managed. By letting the leg prosthesis system 1 cooperate with the prosthesis wearer's body and existing lower extremities, it is possible to imitate the energy-saving way of the human body to perform the movement. For minimum consumption of energy, all joints in the lower extremities are allowed to cooperate, and the remaining stump of the prosthesis wearer can cooperate with the at least one movable joint in the leg prosthesis system. The leg prosthesis system should supplement the prosthesis wearer and should preferably, but not restrictively, be controlled by him or her. [0068] With a leg prosthesis system 1 and/or a method according to the invention, the disconnectable active drive unit 4, 4 ′ of a knee joint or ankle joint makes it possible for the system and the method to use a combination of active and passive operation. The control system 15 can select the optimal method of performing a movement. The knee joint 2 can be active while the ankle joint 3 is passive and vice versa. For example, the knee joint 2 can perform the swinging movement in the swing phase using only the passive brake unit 2 while the ankle joint 3 uses its active drive unit for dorsal flexion of the foot in order to create extra ground clearance. [0069] FIG. 10 and FIG. 11 are side views in cross-section of a knee joint which, for instance, may be included in the leg prosthesis system 1. The socket 13 is connected to the movable knee joint 2 which in turn is connected to a hydraulic piston 9 via a link arm 10. FIG. 11 shows how the piston 9 is moved when the knee joint is angled. FIG. 10 shows a knee joint 2 according to the invention in its active state with a drive unit 4, a brake unit 5 and a control system 15. In this embodiment, the brake unit 5 involves throttling of the hydraulic oil which provides braking/dampening of the movement of the joint. The battery 11 drives the hydraulic pump 6 of the drive unit 4 via a motor (not shown) for operating the valve 8 of the brake unit 5. The battery 11 also drives the control system 15 and transducers and sensors (not shown) of the leg prosthesis system 1. The control system 15 in turn controls the drive unit 4 and the brake unit 5 and receives input data from transducers and sensors; in addition the control system 15 coordinates the movements of the knee joint 2 and the ankle joint 3. [0070] For activation of the drive unit 4, according to FIG. 10, the hydraulic pump 6 is started, the pressure increases on one side of the pump 6 and, via one of the ducts which open adjacent to the spring 17, the valve cone 7 is pressed aside and the duct system of the drive unit will communicate with the cylinder 16 where the piston 9 works and thus the pump 6 actuates the piston 9 in one or the other direction. When the active drive unit 4 is activated, the valve 8 in the passive brake unit 5 should be completely closed to be able to use the maximum efficiency of the hydraulic pump 6. The active drive unit can drive the knee joint 2 in both directions, in the direction towards a straightened knee joint and in the direction to bend the knee joint. In FIG. 10, the active drive unit acts to straighten the knee joint 2. When the piston 9 is moved in the cylinder 16, it acts on the link arm 10 which in turn acts on the knee joint 2 to perform a movement. Alternative types of driving and motors can be used for the leg prosthesis system other than those mentioned above. [0071] According to FIG. 11 the drive unit 4 is disconnected by the hydraulic pump 6 being switched off. The pressure decreases and the spring 17 presses the valve cone 7 back to its rest position, that is the valve cone 7 closes the ducts to the drive unit 4. The brake unit 5 is activated when the drive unit 4 is disconnected. A movement of the knee joint 2 actuates the piston 9 via the link arm 10, the hydraulic oil in the cylinder 16 is pressed through the valve 8 of the brake unit 5 and the degree of braking/dampening can be adjusted by varying the opening degree of the valve 8. The braking can be varied in brake force and can be varied from a completely unbraked (freely swinging) to a completely braked (locked) knee joint 2. [0072] The foot 14 with the ankle joint 3 according to FIG. 12 is shown in its active state and functions similarly to the knee joint according to FIG. 10 and FIG. 11. To activate the drive unit, the hydraulic pump 6 ′ is started, the pressure is increased on one side of the pump 6 ′ and via one of the ducts which open adjacent to the spring 17 ′, the valve cone 7 ′ is pressed aside and the duct system of the drive unit will communicate with the cylinder 16 ′ where the piston 9 ′ works. In this manner, the pump 6 ′ actuates the piston 9 ′ in one or the other direction. When the active drive unit 4 ′ is activated, the valve 8 ′ in the passive brake unit 5 ′ should be completely closed to be able to use the maximum efficiency of the hydraulic pump 6 ′. The piston 9 ′ actuates the link arm 10 ′ which in turn actuates the ankle joint 3 to perform a movement of the foot 14 relative to the interconnecting element 12. The drive unit 4 ′ is disconnected by the hydraulic pump 6 ′ being switched off. The pressure decreases and the spring 17 ′ presses the valve cone 7 ′ back to its rest position, that is the valve cone 7 ′ closes the ducts to the drive unit 4 ′. The brake unit 5 ′ in FIG. 12 is then activated; in normal working conditions the hydraulic pump 6 ′ is then switched off. [0073] A movement of the ankle joint 3 in the passive state actuates the piston 9 ′ via the link arm 10 ′, the hydraulic oil in the cylinder 16 ′ is pressed through the valve 8 ′ of the brake unit 5 ′ and the brake force can be adjusted by varying the opening degree of the valve 8 ′. The braking can be varied in brake force and can be varied from a completely unbraked (freely swinging) to a fully braked (locked) ankle joint 3. [0074] If the leg prosthesis system 1 merely comprises a foot prosthesis 3 according to the invention, for instance at an amputation level below the knee joint, the foot prosthesis still needs a battery 11 ′ and a control unit 15 ′ which may then be arranged, for instance, around the interconnecting element or on a leg-enclosing socket. [0075] It goes without saying that the invention should not be considered limited to the embodiments described above and illustrated in the drawings, with the described variants and alternatives, and can be modified additionally in various ways within the scope of the appended claims. | Summary: A lower limb prosthesis system and a method of controlling the prosthesis system to replace a missing lower extremity of an individual and perform a gait cycle are disclosed. The prosthesis system has a controller, one or more sensors, a prosthetic foot, and a movable ankle joint member coupled to the prosthetic foot. The movable ankle joint member comprises a hydraulic damping system that provides the ankle joint member damping resistance. The controller varies the damping resistance by providing volumetric flow control to the hydraulic damping system based on sensor data. In one embodiment, the hydraulic damping system comprises a hydraulic piston cylinder assembly, hydraulic fluid, and a valve to regulate the fluid. In one embodiment, the controller alters the damping resistance by modulating the valve to vary the hydraulic fluid flow within the hydraulic piston cylinder assembly of the movable ankle joint member based on sensor data. | 7,366 | 189 | big_patent | en |
Summarize: TECHNICAL BACKGROUND [0001] The present invention relates to intraocular lenses implanted into the eye for the correction of vision. When a lens is implanted in an eye that contains a natural crystalline lens, the implanted lens is a phakic lens. A phakic lens may be implanted into the anterior chamber, the area behind the cornea and in front of the iris, or into the posterior chamber that is behind the iris. A posterior chamber phakic refractive lens (PRL) is surgically implanted behind the iris and in front of the human natural crystalline lens for correcting ametropia or refractive errors, such as myopia and hyperopia. Implantation of a phakic refractive lens is the only reversible surgical procedure for correcting severe refractive errors in myopic and hyperopic patients. A number of possible complications have slowed the acceptance of this procedure. They are (1) intraocular pressure (IOP) elevation; (2) cataract induction; and (3) iris pigment dispersion. These complications have been linked to lens designs that are permanently fixed in the eye through attachment to anatomical structures such as the ciliary sulcus and iris. [0002] A floating phakic refractive lens has been designed which preserves eye dynamics and greatly reduces the risk of these complications, especially compared to other phakic refractive lens designs. The floating design allows aqueous flow in the eye to reduce or eliminate the risk of intraocular pressure rise and reduces the chance of contact of the refractive lens with the natural crystalline lens that could induce a cataract or forced connection to the iris that causes iris pigment dispersion. A floating lens design has solved these serious problems by its ability to move with the dynamic changes in the eye, such as accommodation, but this freedom of movement can lead to decentration away from the optical center of the eye within the iris opening (pupil). Decentration can be associated with a rare but potentially serious complication, movement of the lens past the zonules and into the vitreous cavity behind the natural crystalline lens. The zonules are fibers connecting the ciliary process of the eye to the natural crystalline lens. In some people, especially those with very high degree of myopia or hyperopia, the zonules may become weakened and/or detached. If one side of a decentered floating phakic refractive lens, the tip of the haptic member, comes to rest on the zonules, loss of zonule integrity could allow the resting lens to slip through the gap. An additional surgical manipulation would then be required to retrieve the phakic refractive lens. [0003] An improved method for centering the phakic refractive lens that preserves the benefits of that design and does not depend on problematic permanent fixation is needed. [0004] There are a number of patents describing the posterior chamber phakic refractive lens concept and specific lens designs. U.S. Pat. No. 4,585,456, Blackmore, issued Apr. 29, 1986, discloses a phakic intraocular lens composed of flexible materials which is positioned against the natural lens of the eye and is held in place immediately adjacent to the natural lens and the ciliary sulcus. The lens is fixed in place, rather than floating. It assumes that stable centration is achieved by fixing the position of the lens through direct and constant contact with the tissues and structures of the eye. Intraocular pressure elevation and cataract formation are complications from such a lens design. These complications are documented by Fechner, et al, in the Journal of Cataract and Refractive Surgery, March 1996, Volume 22, pages 178-81. Even in a fixed lens that has a length matched to the diameter of the eye, which is difficult to achieve, the lens will eventually contact the natural lens, resulting in a subcapsular cataract. This is because the natural lens grows throughout life and will eventually press against the fixed refractive lens. [0005] Other patents describe different ways of reducing intraocular pressure elevation and avoiding cataract formation by phakic refractive lens designs and their fixation mechanisms. Fedorov, in U.S. Pat. No. 5,480,428, issued Jan. 2, 1996, discloses a novel phakic lens design with an opening through the center of the optic body. This open hole allows aqueous humor to flow through the lens body, from its source in the posterior chamber to the anterior chamber of the eye. The hole is designed to restore aqueous flow and reduce intraocular pressure that builds when the lens body blocks the iris opening. The hole was also found to reduce the optical performance of the lens. Fedorov, in U.S. Pat. No. 5,258,025, issued Nov. 2, 1993, discloses that post-operative inflammation, caused by the supporting element's contact with ocular tissue, is prevented by moving supporting elements to the periphery of the phakic lens. The patent teaches that the Zinn's zonules are strong enough to hold the supporting elements in place without causing inflammation. Feingold, in U.S. Pat. No. 5, 913,898, issued on Jun. 22, 1999, discloses an improvement of the Fedorov posterior chamber phakic refractive lens design with the supporting elements placed in the ciliary sulcus; the lens includes a hole in the center of the optic for equalizing pressure in the eye. These fixed designs are wide and span the inner diameter of the posterior chamber, effectively blocking the aqueous flow from the anterior chamber where it should exit the eye. The natural pressure equalizing mechanism has been compromised, effectively creating conditions that could lead to glaucoma. A hole in the lens allows the aqueous flow to be re-established. A number of later patent applications, such as Patel, U.S. Published Application 2003/0204253, published Oct. 30, 2003, and Bogaert, U.S. Published Application 2005/0149184, published Dec. 14, 2004, disclose holes or penetrating channels in the phakic lens body for the purpose of allowing aqueous flow that are said to prevent intraocular pressure increases. These are posterior chamber phakic lens designs that are fixed in place and use a hole in the optic body to reduce intraocular pressure that may lead to glaucoma. [0006] Kawamorita et al, in “Fluid Dynamics Simulation of Aqueous Humor in a Posterior-Chamber Phakic Intraocular Lens with a Central Perforation”, Graefes Arch Clin Exp Ophthalmol (2012), Volume 250: 935-939, discuss a computer model of the flow of aqueous humor through a hole in the center of the optic of a posterior chamber phakic refractive lens. The authors indicate that the hole was created to allow increased aqueous flow between the natural lens and phakic implanted lens and to equalize pressures in the eye. The analysis shows that aqueous humor is channeled through the center hole at a higher rate than the flow around the lens body. The analysis also showed that the flow velocity between the anterior surface of the natural crystalline lens and the posterior surface of the posterior chamber phakic lens is increased relative to the situation with the same phakic lens design without a center hole. In this case, the lens design includes fixation of the haptic members in the ciliary sulcus. In other words, the lens is not a floating design. [0007] The floating phakic refractive lens design is disclosed in Valyunin, et al, U.S. Pat. No. 6,015,435, issued Jan. 18, 2000, and U.S. Pat. No. 6,428,574, issued Aug. 6, 2002. The floating lens design has an annular ring around the optic area, in the case of a lens for the correction of myopia, or a protruding optic, in the case of a lens designed for treatment of hyperopia, that comes in contact with the edge of the iris, the iris thereby applying a centering force to the lens. A haptic member is attached to the optic that is designed to prevent the optic from being grossly decentered away from the pupil. The haptic is designed with a curvature substantially equivalent to the curvature of the natural lens (see FIG. 1 and FIG. 6 of Valyunin). This floating lens design works with the iris to channel aqueous flow around the lens to reduce the risk of intraocular pressure rise and help to maintain a gap between the implant and the natural lens. The floating behavior of the lens is further assured by matching the material properties of the lens to the aqueous medium of the eye, disclosed in U.S. Pat. No. 6,706,066, Zhou et al, issued Mar. 16, 2004. The specific gravity and mass per unit surface area of the phakic lens material are combined with the phakic lens design to cause the lens to float in the eye and allow the eye dynamics to help maintain the position and centration of the phakic refractive lens. Koivula, et al, in Opthalmology (2007), Volume 114, pages 2031-2037, used optical coherence tomography imaging to show that such a lens moves with the natural eye dynamics to allow a normal aqueous humor flow inside the posterior chamber. [0008] Perez-Cambrodi, et. al., in the Journal of Optometry Jul. 2012, Volume 5, pages 121-130, found, in an analysis of a series of patients implanted with floating phakic refractive lens, that there was contact between one of the haptic members and the zonules in some cases. Positional analysis indicated a trend toward nasal decentration. This did not have a significant effect on the performance of the phakic refractive lens, but continued contact of the phakic refractive lens with the zonules could eventually lead to the complication of zonular dehiscence and dislocation of the lens toward the vitreous chamber (the back of the eye near the retina). Adding an additional design feature that could preserve a floating feature could have significant patient benefits while improving centration of the lens would be a significant improvement, further reducing risks associated with an anatomically compatible phakic refractive lens designed to restore emmetropia in myopic or hyperopic human eyes. BRIEF SUMMARY [0009] The current invention provides a posterior chamber floating phakic refractive lens, with particular lens design and materials, that may be placed in the posterior chamber of the human eye for correction of refractive errors. This invention also provides a phakic refractive lens that can float in aqueous humor and that is flexible and soft. The floating action of the thin, rectangular, buoyant lens will help preserve the eye dynamics so that risks of cataract induction of the human crystalline lens, iris pigment dispersion and intraocular pressure increase are significantly reduced as complications of the implantation of a phakic refractive lens. The present invention uses a small center hole added to the lens optic to cause aqueous flowing through the hole to exert a centering force to the floating phakic refractive lens while preserving optical function. A floating lens design is the only design able to make use of aqueous medium flowing through a central hole to help achieve a stable centration of the optic and to help move the optic body into the pupillary space, maintaining the gap between the phakic refractive lens and the natural crystalline lens. [0010] As used herein, all percentages and ratios are “by weight”, unless otherwise specified. In addition, all patents, patent applications and publications cited herein are incorporated herein by referenced. DESCRIPTION OF THE DRAWINGS [0011] FIG. 1 is a cut-away view of the eye showing the positioning of the lens of the present invention. The cornea, the front surface of the eye, is item 1. The iris is seen at item 2. The natural crystalline lens is item 3. The posterior chamber is item 5 and the anterior chamber of the eye is item 7. The phakic intraocular lens of the present invention is item 6. [0012] FIG. 2 includes a top view ( FIG. 2 a ), side view ( FIG. 2 b ) and perspective view ( FIG. 2 c ) of the lens of the present invention intended for treatment of hyperopia, including arrows that show aqueous flow through the central hole (item 9 ). In these figures, item 10 is the optic or optical body and item 11 is the haptic. [0013] FIG. 3 includes a top view ( FIG. 3 a ), side view ( FIG. 3 b ) and a perspective view ( FIG. 3 c ) of the lens of the present invention intended for treatment of myopia, including arrows that show aqueous flow through the central hole. [0014] FIG. 4 includes a top view ( FIG. 4 b ) and side view ( FIG. 4 a ) of a prior art fixed position intraocular lens where haptic members fix the lens position through contact with tissues of the eye. [0015] FIG. 5 includes top views of three prior art fixed position intraocular lenses. Item 7 indicates a feature that fixes the lens position through attachment to the iris. Item 8 indicates a lens design feature that fixes the lens position in the eye through fixation with the tissue of the ciliary sulcus. [0016] FIG. 6 includes a top view ( FIG. 6 a ), side view ( FIG. 6 b ) and perspective view ( FIG. 6 c ) of a lens of the present floating lens invention designed for the treatment of myopia that includes some description of the design. DETAILED DESCRIPTION [0017] The present invention is a specific lens design improvement meant to solve problems with prior art phakic refractive lens designs by using the existing dynamics of the eye, including the ever-present aqueous flow and accommodation mechanisms, to assist with centering the floating phakic refractive lens, making it a more effective treatment for refractive errors. A floating phakic refractive lens is made with materials that have a specific gravity (e.g., from about 0.9 to about 1.2 g/cm 3 ) near the specific gravity of the aqueous humor of the eye. This will allow buoyant floating of the lens, particularly because the design, disclosed in U.S. Pat. No. 6,015,435, Valyunin et. al., issued Jan. 18, 2000, incorporated herein by reference, includes one or more thin haptic members extending from the optic that are in a rectangular airfoil-like shape similar to the shape of a modern, rectangular parachute. This gives the top view of the lens a generally rectangular shape (see FIG. 2 a ). Continuing the parachute analogy, the history of parachute design shows that violent swaying and oscillating motions were a characteristic of early parachutes without a central vent hole. The flowing surrounding medium (whether air or water) behind the center canopy, which would correspond to the optic zone for a floating phakic refractive lens, could spill around alternating sides of the canopy, causing severe oscillatory movement. A vent hole in the center of the canopy or optic produces a vertical stream of fluid that suppresses eddy currents and stabilizes the position of the parachute or parachute-like lens. Experience has shown that a circular parachute with a central vent hole is more stable than a parachute without the hole but positional stability and control of final landing position are still not optimal with a purely round canopy. Improvements in stability and precise control of parachute movement have been made by moving to rectangular parachute designs. [0018] The rectangular design helps a parachute float in the surrounding medium in a more stable fashion than a purely circular design; fluid that flows around the edges of the rectangle help prevent destabilizing eddy currents. The rectangular design can be maneuvered more precisely as it responds smoothly to changes in flow direction of the surrounding medium that could be affected by the parachutist. A shape with a high aspect ratio, defined as a design which is longer than it is wide, is known in nature and aeronautical science to give an increased amount of lift. Using an aspect ratio in a range between about 1.4 and about 2.0 (length divided by width) results in a stable, maneuverable parachute that is preferred for precision formation parachute flying and landing. The lens design disclosed in U.S. Pat. No. 6,015,435 (Valyunin et. al.) and U.S. Pat. No. 6,706,066 (Zhou et. al.), both incorporated by reference herein, has an aspect ratio in that range and bears a resemblance to such a rectangular parachute but the preferred embodiment of the invention in those patents did not include a hole in the center of the optic body as it was not needed for a floating lens design to equalize pressure in the eye or to prevent a suction force that caused the phakic lens to attach to the natural crystalline lens, as in the case of the Fedorov design disclosed in U.S. Pat. No. 5,258,025. See also U.S. Published Patent Application 2007/0162118, Rozakis et al, published Jul. 12, 2007, incorporated herein by reference. [0019] The phakic refractive lens design of the present invention floats in an aqueous medium that is not static but flowing around the lens body, like a parachute in flight, with a flow from the choroid in the posterior chamber, through the pupil and into the anterior chamber. The aspect ratio of the floating lens design is between about 1.4 and about 2.0, similar to the stable, maneuverable parachute designs used for precision formation flying, even under windy conditions. The floating behavior is further enhanced by the material used to make the thin, rectangular lens. The material is designed to have a specific gravity close to the surrounding aqueous medium so that the lens can be buoyant and float within the aqueous medium (e.g., a specific gravity between about 0.9 and about 1.2 g/cm 3 ). The lenses of the present application can be made from materials which can be hydrophilic or hydrophobic, have the desired optical properties, and are foldable with quick shape recovery, and include, for example, silicone polymers, poly(acrylates), poly(methacrylates), hydrogels, proteins, collagens, copolymers, and mixtures thereof. The materials typically have a hardness of from about 20 to about 60 Shore A. [0020] The lens herein includes a hole in its central portion. The hole is generally from about 0.2 to about 0.5 mm in diameter, such as about 0.35 mm in diameter. The hole is located in the central portion of the optic body; it can be located at the true center of the optic, or the center of the hole can be located within a 0.2 mm diameter circle defined from the true center of the optic body. Typically, only a single hole is used. Multiple holes raise the risk of reduced optical quality. The overall length of the lens of the present invention (i.e., from haptics end to haptics end is from about 10 mm and about 12.5 mm). [0021] With the lens floating in the posterior chamber of the eye, design features have been added to encourage movement of the lens towards the center of the pupil. The lens design with a negative curvature for the treatment of myopia includes a raised ring around the optic that has a smooth, rounded shape. The lens with a positive curvature on the anterior side has an optic zone that extends out with a gentle curve from the level of the haptic members. These protrusions will be captured by the edge of the iris opening. When the eye is exposed to light and the iris closes to adjust the amount of light (i.e. the pupil becomes smaller in diameter), the lens body is moved towards the center of the pupil. In this way, the optic area is designed to be captured by the iris, helping to keep the lens floating towards the optical center of the eye. [0022] A weakness of depending solely on this centration mechanism is that the pupil of some individuals may open to 6 mm or more in diameter in the dark or when sleeping. The lens body could be allowed to move towards one of the zonules, the fibers suspending the natural lens in the eye. Analysis of the phakic refractive lens design in the light of studies on the aqueous flow within the eye suggests that positional stability and centration of the lens could be improved by adding another feature that used the existing fluid dynamics of the eye. Richard F. Brubaker, in his Friedenwald Lecture, “Flow of Aqueous Humor in Humans”, teaches that aqueous homor is continuously being transported into the posterior chamber of the eye through ciliary epithelial cells. The aqueous humor flows through the pupil into the anterior chamber at an approximate mean rate of 2.75±0.63 μl/minute in normal human subjects, circulates around the anterior chamber, and exits through various mechanisms. Kawamorita et. al. investigated the fluid dynamic characteristics of aqueous humor in a phakic refractive lens with a hole in the center of the optical body using computational fluid dynamics. Using the aqueous humor flow values described in Brubaker, they found a significant increase in flow velocity between the anterior surface of the natural lens and the center of the posterior surface of the refractive lens around the hole, with a sharp flow rate increase of 0.15 mm/second in the center of the hole. Trajectory analysis showed aqueous humor flow through the hole and into the anterior chamber. Practical experience with a hose nozzle shows that water accelerates when pushed through a narrow hole. Consideration of the eye's aqueous flow dynamics described by Brubaker and the computer model of aqueous flow through a center hole of a phakic refractive lens by Kawamorita et al suggests that a central hole in the optic body of a floating phakic refractive lens could be a centering feature. A central vent hole added to the floating lens design, as it was to parachute designs, improves the centration and position stability of the lens in the flowing aqueous medium. Another benefit of this improved design element is that the slight acceleration of the flow through the hole would tend to move or lift a floating lens in the direction of the center of the pupil, like a parachute opening in a column of air. This could possibly increase or help maintain the gap between the floating lens and the natural crystalline lens, further reducing the risk of the floating lens touching the natural lens. In addition, this gap is maintained by the fact that the posterior surface curvature of the present lens is similar to the anterior curvature of the natural human lens. The floating phakic refractive lens has been designed to reduce the risk for complications such as increased intraocular pressure, pigment dispersion, secondary cataract, and loss of endothelial cells compared to other phakic refractive lens designs. The addition of the hole in the optical body of the present invention to help centration of the refractive lens is a significant improvement for a safe and effective treatment for refractive errors, such as myopia and hyperopia, that affect millions of people. EXAMPLES [0023] The following examples are given for the purpose of illustrating the present invention and are not intended to be limiting thereof. Example 1 Floating Silicone Phakic Refractive Lens (Prior Art) [0024] A small amount of a two-part silicone material with a specific gravity between 0.95 and 1.05 is mixed at a 10:1 ratio and placed in a metal mold. The mold is clamped shut and placed in a curing oven at 120° C. for 70 minutes. The mold is cooled to room temperature, opened, and the phakic lens is carefully removed. The phakic lens has the configuration and dimensions of the lens in FIG. 1. [0025] The lens is placed in a container with water and the lens can be seen to float on the top of the water. If forced under the water surface, the lens remains floating under the surface. If a stream of moving water is caused to flow under the floating lens, the lens returns to the surface. The lens movement is uncontrolled, with occasional movements towards the side of the container or exaggerated rocking movements. Example 2 Floating Silicone Phakic Refractive Lens With Center hole [0026] A small amount of a two-part silicone material with a specific gravity of 0.95 to 1.05 is mixed at a 10:1 ratio and placed in a metal mold. The mold is clamped shut and placed in a curing oven at 120° C. for 70 minutes. The mold is cooled to room temperature, opened, and the phakic lens is carefully removed. A hole with a diameter of approximately 0.4 mm is made in the center of the optic. The phakic lens has the configuration and dimensions of the lens in FIG. 6. [0027] The lens is placed in a container with water and the lens can be seen to float on the top of the water. If forced under the water surface, the lens remains floating under the surface. If a stream of moving water is caused to flow under the floating lens, the lens returns to the surface. The lens movement is more controlled than the lens with no hole in the center; the lens rises vertically and the lens orientation is conserved. Example 3 Floating Silicone Phakic Refractive Lens With Center Hole [0028] The phakic refractive lens of Example 2 is evaluated for optical performance and compared to the optical performance of the lens of Example 1 that has no hole in the center of the optic. A commonly used method for evaluating optical performance is to place the lens on an optical bench with a collimator, objective lens, and a US Air Force 1951 Target or similar target image. The ability of the test lens to completely and clearly resolve an image pattern such as a group of closely spaced lines or bars is evaluated as a measure of resolution efficiency and imaging quality. Another measure of imaging quality and optical performance is the modulation transfer function (MTF). It is found that when the center hole is between 0.2 and 0.5 mm in diameter, there is no significant difference in optical performance between the lenses of Example 1 and Example 2. | Summary: An improved self-centering phakic refractive lens is disclosed. The lens floats freely in the posterior chamber of the eye and corrects vision of the patient but also prevents buildup of intraocular pressure, cataract induction and iris pigment dispersion. The lens comprises an optical body, haptic members which extend outward from the optical body, and a small hole in the approximate center of the optical body for the purpose of allowing aqueous humor to flow through that hole. The lens is designed such that its posterior surface conforms to the shape of the anterior surface of the patient's natural crystalline lens. | 6,125 | 137 | big_patent | en |
Write a title and summarize: The Gran Chaco ecoregion, a hotspot for Chagas and other neglected tropical diseases, is home to >20 indigenous peoples. Our objective was to identify the main ecological and sociodemographic determinants of house infestation and abundance of Triatoma infestans in traditional Qom populations including a Creole minority in Pampa del Indio, northeastern Argentina. A cross-sectional survey determined house infestation by timed-manual searches with a dislodging aerosol in 386 inhabited houses and administered questionnaires on selected variables before full-coverage insecticide spraying and annual vector surveillance. We fitted generalized linear models to two global models of domestic infestation and bug abundance, and estimated coefficients via multimodel inference with model averaging. Most Qom households were larger and lived in small-sized, recently-built, precarious houses with fewer peridomestic structures, and fewer livestock and poultry than Creoles’. Qom households had lower educational level and unexpectedly high residential mobility. House infestation (31. 9%) was much lower than expected from lack of recent insecticide spraying campaigns and was spatially aggregated. Nearly half of the infested houses examined had infected vectors. Qom households had higher prevalence of domestic infestation (29. 2%) than Creoles’ (10. 0%), although there is large uncertainty around the adjusted OR. Factors with high relative importance for domestic infestation and/or bug abundance were refuge availability, distance to the nearest infested house, domestic insecticide use, indoor presence of poultry, residential overcrowding, and household educational level. Our study highlights the importance of sociodemographic determinants of domestic infestation such as overcrowding, education and proximity to the nearest infested house, and corroborates the role of refuge availability, domestic use of insecticides and household size. These factors may be used for designing improved interventions for sustainable disease control and risk stratification. Housing instability, household mobility and migration patterns are key to understanding the process of house (re) infestation in the Gran Chaco. The strong association between neglected tropical diseases (NTDs), poverty and particular combinations of ecological, social, political and economic determinants explains the occurrence of global hotspots of NTDs [1]. One of such hotspots occurs in the Gran Chaco ecoregion in South America, where the prevalence rates of geohelminthic infections and Chagas disease are very high [1]. Chagas disease, caused by Trypanosoma cruzi, is considered the main regional vector-borne disease in terms of disease burden and affects 8–10 million people in Latin America [2]. Triatoma infestans, the main vector in the Southern Cone countries and southern Peru, has been the target of an insecticide-based regional elimination program that interrupted the transmission of human T. cruzi infection by T. infestans in various countries [2–4]. Progress in the Gran Chaco lagged behind and vector-mediated transmission of T. cruzi still occurs albeit at lower incidence levels than 20 years ago [5–8]. The Gran Chaco is home to more than 20 ethnic groups [9]. Indigenous populations usually are among the most marginalized groups, with more precarious health and living conditions than other peoples [10–12]. Indigenous communities of the Gran Chaco showed high seroprevalence of human T. cruzi infection [13–18]. One of the most numerous ethnic groups in this region is the Qom (Toba) people [19]. Qom households were exposed to a greater risk of T. cruzi infection than Creole households in a well-defined rural section of Pampa del Indio (Argentine Chaco) mainly inhabited by Creoles (denominated Area I), but there were large heterogeneities between and within ethnic groups [20,21]. Further studies on the ecological, biological and social (eco-bio-social) determinants of vector-borne diseases are needed [22], more so in the case of vulnerable indigenous populations affected by Chagas disease and other NTDs. The main identified determinants of house infestation with the major domestic vectors of T. cruzi (T. infestans, Rhodnius prolixus, Panstrongylus megistus, and Triatoma dimidiata) include housing construction characteristics that create refuges for the bugs to hide in (e. g., cracks in walls, thatched roofs, precarious peridomestic structures); the presence and number of human and domestic animal hosts (dogs, chickens) that serve as bloodmeal sources, and little or no domestic application of insecticides by house residents [20,23–32]. These factors are the expression of various underlying processes that ultimately create conditions that facilitate house infestation and T. cruzi transmission [33]. A full understanding of complex systems [34] involving infectious diseases requires more integrative approaches such as the ecosystem approach to human health (ecohealth) [35], which gives proper attention to eco-bio-social factors and their eventual interactions. However, very few studies have explicitly addressed these factors simultaneously in relation to Chagas disease [31,36,37]. This limited knowledge curtails our ability to design and implement innovative vector and disease control strategies adapted to resource-constrained settings. The current study therefore addressed traditional ecological determinants and selected sociodemographic factors related to poverty and ethnicity. As part of a longitudinal study on the eco-epidemiology and control of Chagas disease in northeastern Argentina, we expanded the scope and geographic scale of our previous studies [20,21,37] conducted in Area I of Pampa del Indio to focus on Qom communities living in ancestral territories which also included a Creole minority (denominated Area III). The living conditions of Qom households most likely differed substantially from those of Creoles, and their association with house infestation has not been investigated at a sufficiently large spatial scale. The objective of the current study was to identify the main ecological and sociodemographic determinants of domestic infestation and abundance of T. infestans (two surrogate indices for transmission risk) in Area III, where Qom communities predominated, using generalized linear models in a multimodel inference frame with model averaging. In addition to the above-mentioned factors known to be closely associated with house infestation in multiple settings, we examined the effects of distance to the nearest infested house, residential overcrowding, household education level, wealth indicators, and preventive practices. The first two factors were predicted to exert positive effects on domestic infestation and bug abundance whereas the remaining factors were expected to exert negative effects. We also re-examined whether ethnic background modified both response variables when other relevant risk factors were accounted for. Our study highlights the relevance of various ecological and sociodemographic factors whose effects have not been investigated simultaneously, and provides guidance on improved control interventions specifically adapted to the Gran Chaco. Field work was conducted in a rural section (95 km2) of Pampa del Indio municipality (25° 55’S 56° 58’W), Chaco province, Argentina (Fig. 1). The municipality was inhabited by approximately 22,000 people by late 2013, and 45% of residents belonged to the Qom ethnic group according to local municipal authorities. Official decennial census records in 2001 and 2010 indicated that the population of Pampa del Indio municipality increased remarkably from 11,558 to about 18,000 people, respectively (annual population growth rate, 4. 9%). The climate, landscape and demographic features of a contiguous section of the municipality inhabited mainly by Creole households were described elsewhere [20,21]. The last insecticide spraying campaign conducted in the municipality occurred in 1997–1998 according to the Chagas disease control program from Chaco province. Selection of the study area took into account the lack of recent history of community-wide insecticide spraying; preliminary evidence of house infestation ranging from 30 to 40%; the predominance of indigenous households; and the presence of at least 350 adjacent households in order to achieve a sufficiently large study base for statistical inference. A household is defined as all the people who occupy a housing unit including the related and nonrelated family members [38]. A house compound was defined as the set of domicile (i. e., an independent structure used as human sleeping quarters, S1 Fig.), patio and other structures included within the peridomestic area (kitchens, storerooms, latrines, corrals, chicken coops and chicken nests (“nidero”), ovens, trees where chickens roosted, others) as illustrated elsewhere [20]. House compounds sometimes had more than one domicile used as sleeping quarters by related family (S1 Fig.). Before initiating field operations local key actors were interviewed to gather background data that may allow a better assessment of the preintervention situation; discuss the initial and long-term goals of the research program (see below); and assist the interpretation of the study outcomes. Local key actors included the mayor, health and education authorities and other personnel, rural health-care workers and school teachers, representatives of third-sector organizations, and community leaders. The stated long-term goals of the research program were to interrupt the human transmission of T. cruzi through intensified vector control, human diagnosis and treatment, and to promote long-term sustainability of disease control efforts through local empowerment. A cross-sectional survey aimed at enumerating all house compounds in the area and assessing house infestation was conducted in October 14–31,2008. The study area included seven villages with 407 inhabited houses, 19 abandoned dwellings and 17 public buildings (4 primary schools, 1 health-care post, 6 churches and 6 community centers) (Fig. 1). One member of the research team explained to each householder the aims of the survey and requested permission to access their premises and identify the house with a numbered aluminum plate. The location of each house was georeferenced with a GPS receiver (Garmin Legend). Householders were asked for the presence of triatomines within their premises after showing them dry specimens of T. infestans, Triatoma sordida and other Reduviidae to prevent confusion with other insects; from these reports we derived the index “householders’ notification of house infestation”. All households were provided with a labeled self-sealing plastic bag to contain any triatomine they sighted, and instructed on how to manipulate the bugs safely. This additional source of bugs was denominated “householders’ bug collections”. Householders’ bug collections were only considered if the date and collection site were reported to us. The study protocol was approved by the Dr. Carlos A. Barclay Independent Ethical Committee for Clinical Research, Buenos Aires, Argentina. A total of 386 inhabited houses (94. 9%) were included in the current study of triatomine infestation; 21 houses closed during the survey were not searched for bugs. In all of the 386 study houses the following methods were performed to assess bug infestation: i) inspection by timed-manual searches; ii) collection of bugs that were spotted during insecticide spraying operations; and iii) promotion of householders’ collection of any triatomine they sighted (as explained above). Multiple methods were used as a cross-check of the outcome of timed-manual searches. All domestic and peridomestic sites of the study houses were searched for triatomine bugs (timed-manual collections) by four teams including one supervisor and two or three skilled bug collectors who used 0. 2% tetramethrin (Espacial, Argentina) to dislodge the insects as described [20]. Each domicile and peridomestic site was searched by one person for 15 min. Immediately after the vector survey, vector control personnel sprayed every house with suspension concentrate deltamethrin (K-Othrin, Bayer) or beta-cypermethrin (Sipertrin, Chemotecnica) using standard doses (25 and 50 mg/m2, respectively) and routine procedures [39]. Bugs sighted during insecticide spraying operations were also collected. The collected bugs were stored in plastic bags labeled with the house number and specific bug collection site and were transported to the field laboratory where they were identified taxonomically and counted according to species, stage or sex. Two to six weeks after bug collection, feces of all the third-instar nymphs and older stages that were alive were microscopically examined for infection with T. cruzi at 400× as described [21]. The bugs examined for infection were collected from 72. 8% of the infested houses. This survey was conducted in parallel to the vector survey in October 2008. An adult household member fluent in Spanish was asked for information on the following items: full name of householder (i. e., head of household) [40]; the number of resident people by age class (0–5,6–14, and 15 or more years of age); the number of domestic animals of each type (dogs, cats, chickens, other poultry, goats, pigs, cows, and equines) and their resting places; use of domestic insecticides (type, frequency, purpose); and date of the last insecticide spraying of house premises conducted by vector control personnel or the local hospital or any other third party using manual compression sprayers. Because the study area encompassed traditional Qom communities, assignment of a household to ethnic group was based on whether they spoke Qom language (Qomlaqtaq); participated in traditional Qom organizations; and took into account their physical features. When in doubt, assignments to ethnic group were subsequently checked with local Qom health-care personnel and were corroborated in all cases. Households with a mixed ethnic background were considered to be Qom because they resided in ancestral indigenous territories and fulfilled the above mentioned attributes. A sketch map of the spatial location of all structures in each house compound was performed, and each structure was given a unique code according to its use. We recorded the building materials used in roofs and walls, presence of wall plaster, condition of wall surface, and plaster material. The availability of refuges for bugs was determined visually by a skilled member of the research team and scored in one of five levels ranging from absence to very abundant refuges [20]; only the three top categories were recorded in domiciles. As our knowledge of the study area increased during the vector surveillance phase, additional sociodemographic variables potentially associated with house infestation were taken into consideration and recorded mostly in November 2012: educational level attained by each household member (number of schooling years completed); land ownership (no ownership; individual: the householder owned the land they inhabited; familial: a relative owned the land; communal: the community owned the land which therefore could not be sold); agricultural activities (present and past); monthly public welfare support; household electricity and time since first connection; age of house (years since construction); size of each domicile’s area; source of drinking water; presence of window screens (wire mesh); use of bed nets; and participation in a local social organization. The data collected in 2012 were back-corrected to extant conditions in 2008 (e. g., access to electricity, age of house, agricultural activities). Overall changes in several respects (e. g., drinking water source, domicile’s area, participation in social organizations) were negligible during the four-year period. For some of the back-corrected variables it was possible to assess the validity of the reports. The comparison of domestic area and age of house recorded both in 2009 and 2012 showed minor differences. Land tenure, access to electricity and householders’ reports of time since last insecticide spraying were checked with other local sources of information and whether they were spatially clustered. Comparison between the list of houses sprayed with insecticides in 2006 (identified by the name of the head of household) and the date of the last insecticide spraying each individual household reported to us in 2008 showed either large or perfect agreement in two communities (75% and 100%) and a very low degree of agreement in another community (8%). The recorded data were used to compute household-level surrogate indices for wealth, educational level and overcrowding. The goat-equivalent index represents a small stock unit to quantify the total number of livestock (cows, pigs, goats) and poultry owned by the household in terms of goat biomass. To calculate this index the average weight of each type of animal was considered (cow, 453 kg; pig, 159 kg; goat, 49 kg; chicken, 2. 5 kg) [41]. The household educational level was defined as the mean number of schooling years attained by household members aged 15 years old or more [42]. The overcrowding index was defined as the number of human occupants per sleeping quarter; the presence of 3 or more occupants per room is taken as critical overcrowding [43]. Housing quality (a three-level categorical variable) was represented by the combination of mud walls (versus brick-cement walls) and tarred-cardboard sheets on the roof (versus corrugated metal-sheets); no house had brick-cement walls and tarred-cardboard sheets. As part of annual vector surveillance after community-wide insecticide spraying in October 2008, all extant households in the study area were re-surveyed in August 2009, April 2010 and November 2012, whereas a sample of 86 houses was re-surveyed in December 2011. For each house we recorded its current and previous existence; fate (destruction, movement and construction); destination of moving households and underlying reasons (whenever possible), among other variables. The sociodemographic information was collected at every new house as in the baseline survey. House infestation data only included inhabited houses because no local public building or abandoned house was found to be infested. Similarly, latrines and trees used by chickens were not infested by T. infestans and therefore were not included in the number of peridomestic sites per household. The prevalence of house (or site-specific) infestation by T. infestans was calculated based on the finding of at least one live bug (except eggs) by any of the bug collection methods used (i. e., timed-manual searches, during insecticide spraying operations, and householders’ bug collections) relative to the total number of houses (or sites for each ecotope) inspected. The abundance of triatomine bugs was calculated as the number of live bugs collected per 15 min-person among houses positive by timed-manual searches. If a house compound had more than one domicile, the average domestic bug abundance was calculated as the total number of live triatomines collected per 15 min-person across domiciles divided by the number of domiciles inspected. A matrix of distances to the nearest infested house was calculated using qGIS [44]. Agresti–Coull binomial 95% confidence intervals (95% CIs) were used for infestation prevalence [45]. Householders’ notification of the domestic presence of T. infestans and timed searches of domestic infestation were compared using the kappa index implemented in Stata 12 [46]. Kappa index values greater than 0. 6 may be considered substantial to perfect agreement and values less than 0. 4 represent a poor agreement beyond chance. Risk factor analyses of the presence and relative abundance of T. infestans were restricted to human sleeping quarters because peridomestic infestations were relatively few. Owing to the occurrence of house compounds with more than one domicile (including related family) and that several variables were measured at the household level, in these cases data for all domiciles were pooled to obtain a single observation per compound. Availability of refuges for bugs and age of house were averaged over domiciles within a house compound, and the total domestic area was the sum of each domicile’s area. The number of domestic hosts (dogs or cats and poultry, mostly chickens) used in the analyses (not in the census) only included animals reported to rest or nest inside domiciles. Bivariate logistic and negative binomial regressions on each explanatory variable were performed with domestic infestation and bug abundance as response variables, respectively. Relative bug abundance (RA), labeled in Stata output as' incidence-rate ratios’, and their CIs were calculated from the estimated coefficients (b) of the negative binomial regression as eb. The association between selected explanatory variables and both response variables were tested through multiple logistic and negative binomial regressions, respectively. The global models included 10–12 explanatory variables with complete data selected a priori based on background evidence (e. g., [20,25,30]) and additional hypothesis on the predicted effects of selected sociodemographic determinants as mentioned above. Some variables measured in 2012 (i. e., age of house, electricity, time since last insecticide spraying) had a large number of missing data and therefore were not included in these analyses. We also compared the fit of the negative binomial models for bug abundance with those returned by mixture and two-part models for zero-inflated distributions, and found strong evidence of the superiority of the negative binomial regression model (S2 Text). Two global models were analyzed. The first model included 10 explanatory variables (from 386 households) which described building characteristics (housing quality, refuge availability), domestic host availability (number of persons, number of dogs or cats and presence of poultry indoors), socioeconomic features (ethnicity, goat-equivalent index), household preventive practices (reported insecticide use), peridomestic infestation by T. infestans, and distance to the nearest infested house. The second model included 12 variables (i. e., the 10 variables mentioned before, residential overcrowding and household educational level) recorded at 274 households. Some continuous variables were rescaled in order to give more meaning to the unit of increment of risk estimates: distance to the nearest infested house (one unit every 50 m), household educational level (every 6 years) and the goat-equivalent index (every 10 goats). For comparative purposes we also analyzed the second data set after removing overcrowding and educational level data. On a post hoc basis we investigated the effects of the interactions between ethnicity and every other factor in the global models on both response variables, which proved not to be significant. These terms were added one by one to each global model and tested separately to avoid convergence problems. Potential multicollinearity among explanatory variables was evaluated through the variance inflation factor (VIF) and condition numbers as implemented in Stata 12. The condition numbers were less than 10 and VIF < 2 for all explanatory variables, indicating that the significant correlation found between some pairs of variables (ethnic group with housing quality, refuge availability, goat-equivalents, which had correlation coefficients ranging from 0. 35 to 0. 4) would not cause serious multicollinearity. We used an information theoretic approach and Akaike’s information criterion (AIC) to identify the best-fitting models describing variations in domestic infestation and abundance of T. infestans, given the data collected. Multimodel inference was especially conceived to account for model selection uncertainty; it allows a quantitative ranking of the models and identification of the set of models having best support given the data [47,48]. Because the ratio between the number of parameters and the number of observations (i. e., houses) was less than 40, we used the AIC corrected for small sample size (AICc). Akaike differences (ΔAICc) were calculated for each model as ΔAICc = AICc—AICmin; models with ΔAICc > 2 were considered to have less support than the best model (AICmin), given the data and models analyzed. Several models had substantial support; therefore, we performed multimodel inference through model averaging. The Akaike weight (wi) of a model represents the support or probability of being the “best model”. The relative importance (RI) of each variable is defined as the sum of Akaike weights in each model in which the variable is present; RI takes values from 0 to 1. The overall fit of the logistic models was assessed by the Hosmer-Lemeshow test using the model-averaged coefficients and pooling the data in 10 equal-sized groups. Odds Ratios (ORs) and their 95% confidence intervals were calculated from model-averaged coefficients. Unconditional standard errors were calculated according to equation 4 in [49] with the default option (revised. var = TRUE). The area under the receiver operating curve (ROC) was also calculated; a value of 1 indicates a perfect fit. Sensitivity and specificity were assessed using the observed infestation prevalence of each data set as the cutoff values. The analyses and calculations were performed in R (version 2. 15. 1) [50]. Package MuMIn (version 1. 9. 5) was used for multimodel averaging; ResourceSelection (0. 2–2) for performing the Hosmer-Lemeshow test; and ROCR (version 1. 0–5) for calculating sensitivity, specificity and the area under the ROC curve. The spatial distribution of domestic infestation was assessed through global and local point pattern analyses (PPA) [51]. The former estimates the spatial aggregation of the outcome event across the entire study area whereas the latter detects the location of aggregated events. The spatial distribution of houses was examined to determine whether the potential aggregation of house infestation was influenced by a non-random dispersion of house locations. The global spatial analysis of domestic infestation was performed in Programita using the weighted K-function [52] and random labeling as the null hypothesis (i. e., to assess the spatial distribution of infested houses given the fixed spatial distribution of all houses). The maximum distance considered was 2,000 m (i. e., one-third of the smallest dimension of the area) [51], and the cell size was 40 m. A total of 999 Monte Carlo simulations was performed and the 95% confidence envelope was calculated with the 25th upper and lower simulations. Local spatial aggregation of infestation was tested through the Getis statistic (G*) [53] implemented in PPA [54]. This analysis distinguishes between positive and negative aggregation of events (i. e., infested houses); parameter settings were the same as for the global analysis. The house-to-house census enumerated a total of 2,389 inhabitants in 386 inhabited houses as of October 2008, and 2,356 persons in 445 inhabited households in November 2012. The population included 18. 0% up to five years of age; 27. 7% between 6 and 14 years; and 54. 3% with 15 or more years of age at baseline, and displayed a nearly indistinguishable age distribution in 2012. The mean age was 20. 3 yr whereas the country-wide average was 34. 4 yr. The number of men per 100 women was 109. 2 whereas the average for Argentina was 95. 8 as of 2010 [55]. A summary of the housing and sociodemographic characteristics of the study population by ethnic group is shown in Table 1. The detailed frequency distribution of study variables appears in Tables 2,3 and S1. Qom households comprised 89. 6% of the inhabited houses. Unlike Creoles, most Qom households lived in houses with mud walls and a tarred-cardboard roof, in small-sized (< 30m2), recently-built domiciles with high refuge availability, < 2 peridomestic structures, and with little access to electricity (Table 1). Qom households were larger, more often experienced critical overcrowding (S1 Text), and had lower household educational level than Creoles. The average goat-equivalent index of Creoles (median, 68. 9; first-third quartiles, 5. 7–168. 6) was 69 times larger than that of Qom households (1. 0,0. 1–7. 7). Most Creole households applied insecticides in domestic premises (90. 0%) and had window screens (59. 5%), unlike Qom households (Table 1, S1 Text). Creole households applied high-concentration pyrethroid or carbamate insecticides (27. 5%) much more frequently than Qom households (5. 8%). Householders’ reports indicated that 68. 0% of houses had never been sprayed with insecticides by vector control personnel whereas 21. 5% had been sprayed two years before (Table 3). Local health personnel reportedly sprayed with insecticides 36 and 49 houses, mainly from Cuarta Legua villages in 2000 and 2006, respectively. A key feature of the study population was the very frequent mobility of households to a new house (i. e., housing instability). Of all the inhabited houses enumerated in 2008,20. 2% (78) were demolished or abandoned by 2012, whereas 142 new houses were built over the four-year period, of which only 52 (36. 7%) were present in 2012. Most of the new houses (90. 1%) and the demolished or abandoned ones (98. 7%) belonged to Qom households. Among the latter, movers were relatively disadvantaged compared to nonmovers. On average, movers had a lower goat-equivalent index (median, 0. 5 versus 1. 9); smaller domiciles (24 versus 36 m2); more recently-built houses (69. 2% versus 45. 6%); smaller household size (5. 2 versus 6. 8); and fewer peridomestic sites (1. 3 versus 2. 1). Triatoma infestans was found by timed-manual searches in 108 (28. 0%) of the 386 inhabited houses and in 6. 9% of the 1,744 sites inspected. The median relative abundance was 3 bugs (first-third quartiles, 1–11) per unit of catch effort. Fifth-instar nymphs (24%), males (20%) and females (16%) were the stages most frequently captured. When the finding of bugs by any collection method was considered, the prevalence of house infestation slightly rose to 31. 9% (123 of 386), and was 27. 2% (105 of 386) in domestic sites and 7. 8% (30 of 386) in peridomiciles. A total of 2,362 T. infestans was caught. Triatoma sordida was found in 4. 5% (17 of 386) of houses exclusively in peridomiciles. The contribution of each collection method to detection of domestic infestation is shown in S3 Table. Although the majority of domestic infestations was detected by timed-manual searches, bugs collected by householders (almost exclusively in domestic areas) and during insecticide spraying operations contributed to additional detection of 19 infested houses that timed searches had missed. Detection of domestic infestations by timed-manual searches and householders’ notifications were in poor agreement (kappa index = 0. 3). The ecotopes most frequently infested at site level (as determined by any bug collection method) were domiciles (23. 1%), storerooms (14. 0%), kitchens (6. 3%), chicken nests (6. 0%), and chicken coops (4. 6%) (Fig. 2). The median abundance of T. infestans per unit of catch effort was higher in kitchens, storerooms and chicken nests, but did not differ significantly among ecotopes by negative binomial regression (P > 0. 1 in all cases). The overall prevalence of bug infection with T. cruzi was 25. 0% among 719 live bugs examined, and ranged from 23. 9% (150/628) in domiciles to 33. 0% (30/91) in peridomiciles. Infected bugs were collected in domiciles of 45. 2% (28/62) of the houses with bugs examined for infection, and in peridomiciles of 44. 4% (8/18) of them. Although house infestation occurred across the study area, some communities showed larger domestic infestation than others (range, 12. 2–50. 8%) (Fig. 3, S1 Table). Domestic infestation was significantly aggregated at a global scale at distances ranging from 600 to 2,000 m (S2 Fig.); this means that infested houses were clustered, and on average, for every infested house there was a higher probability of finding another infested house within 600–2,000 m than expected by chance. Local spatial analyses of bug abundance identified clusters of houses located within 40–600 m in some communities (Cuarta Legua, Pampa Chica). The apparent cold spot at the NE angle (Pampa Grande village) was not statistically significant (P > 0. 05). Wall and roof materials were significantly associated with domestic infestation (Table 2). The prevalence of domestic infestation increased significantly with increasing refuge availability levels and numbers of human residents, and declined steadily with increasing age of house and domestic area. Infested domiciles had a significantly smaller area (37. 8 ± 27. 2 m2, n = 84) than non-infested ones (50. 6 ± 39. 9 m2, n = 200; Mann-Whitney test, P < 0. 001). Domestic infestation was higher in houses with at least one infested peridomestic site and fewer peridomestic structures (S1 Table). Domestic bug abundance only was significantly associated with refuge availability, domestic area and the number of dogs. Qom households had a nearly threefold domestic infestation (29. 2%) than Creoles’ (10. 0%), whereas domestic bug abundance was similar between ethnic groups (Table 3). Domestic infestation increased steadily with increasing residential overcrowding from 0% up to 42. 9%, and decreased with increasing household educational level from 30. 1% to 0%. Households reporting insecticide use had a significantly lower infestation (21. 2%) than those that did not (32. 7%). Bug abundance was also significantly associated with residential overcrowding, household educational level, the goat-equivalent index, number of peridomestic sites, land ownership, and access to electricity (Table 3, S1 Table). The signs of the individual effects were the same as for domestic infestation. Households with no window screens had increased domestic infestation and bug abundance, whereas the use of bed nets was inversely associated (S1 Table). No significant association was found between domestic infestation and time since last insecticide spraying (Table 3). Using the first global model including 386 houses, we identified 11 and 6 models with considerable support (ΔAICc < 2) for domestic infestation and bug abundance, respectively. Refuge availability (RI = 1. 00), distance to the nearest infested house (RI = 1. 00–0. 83) and insecticide use (RI = 0. 75–0. 69) were the most important factors (Table 4). Refuge availability exhibited a strong positive association whereas insecticide use and distance to the nearest infested house had a negative one. The presence of poultry indoors (RI = 0. 75) was only moderately and directly associated with domestic bug abundance. The number of people (RI = 0. 73) had moderate importance and marginally positive effects on infestation only, with CIs including the null value. Other factors presented lower RI for both response variables. Ethnicity showed low RI and widely variable CIs. The average logistic model for infestation (Hosmer-Lemeshow test, χ2 = 8. 03; d. f. = 8; P = 0. 43) and the area under the ROC curve (0. 73) indicated a good fit. The model had moderate specificity (0. 62) and sensitivity (0. 71). For the second global model including 274 houses, refuge availability (RI = 1. 00), overcrowding (RI > 0. 98) and distance to the nearest infested house (RI = 0. 78–0. 88) were the most important factors and showed strong to moderate effects, whereas household educational level had moderate importance (RI = 0. 74–0. 68) and rather small negative effects on domestic infestation only (Table 4). The average logistic model for infestation (Hosmer-Lemeshow test χ2 = 3. 66; d. f. = 8; P = 0. 89) and the area under the ROC curve (0. 79) indicated a good fit. This model showed higher specificity (0. 71) and similar sensitivity (0. 72). Removing overcrowding and household educational level data yielded results that were qualitatively similar to those in the first global model (not shown). Additional analyses including only Qom households identified the same set of factors with high and moderate RI in both global models (not shown). Our study documented threats of active vector-borne transmission of T. cruzi in approximately 27% of the households (as determined by the occurrence of domestic infestations), and identified manageable variables that may be targeted for improved interventions and risk stratification. Improving housing quality and living conditions is urgently needed and largely exceeds Chagas disease vector control because housing improvements will impact positively on family health. Reducing the presence of chickens in human sleeping quarters [20,21,25,26,64,67] and applying insecticides in more effective ways when required may contribute to improved vector control. Although these factors are frequently construed as environmental or ecological, the types of housing, land ownership, habits of raising livestock or poultry, frequency of insecticide use and type of preventive practices have historical, sociodemographic, cultural and political roots. The household mobility patterns recorded have serious implications for vector and disease control. In the preintervention context of an infested area under marginal vector control (as in 2008), the mobility of Qom households implied the potential carriage of bugs in their belongings to the new houses, while leaving bugs behind in the rubble of knocked down walls. The recently-built houses represented new habitat patches susceptible to bug invasion and colonization, and therefore decreased the fraction of all houses effectively protected by the long-lasting residual effects of recent insecticide sprays. On the flip side, the processes of house destruction and reconstruction are expected to cause major negative impacts on the local abundance of bugs by increasing bug mortality and dispersal. The mobility of some indigenous populations may pose special challenges to traditional housing improvement programs relying on stable settlement and secure land tenure. More knowledge of the drivers of household mobility, migration and the desired types of housing of Qom and other indigenous peoples which had a nomadic or seminomadic tradition are needed. The design of Chagas disease prevention programs and other health interventions directed to indigenous populations should address their specific needs and beliefs [80,81]. Improving housing quality in isolation, while traditional agricultural activities continue in decline and other sources of local employment are rare, may not stop the rural-to-peri-urban exodus across ethnic groups. The links between household educational levels and domestic infestation require more elaboration and specific research on the mechanisms involved. This area offers new opportunities for innovative interventions through health education and promotion workshops [82] that include, but are not restricted to, community-based vector and disease surveillance, control and treatment. Better access to formal education may also contribute directly and indirectly to primary and secondary disease prevention (e. g., by increasing awareness of treatment opportunities). The large fraction of Qom and Creole households who managed to keep their premises free from triatomine bugs using the scarce means available to them holds promise for further improvements with a modest investment of resources. Households performing good practices of vector control may contribute as agents of change to further reduce infestation and transmission risks in community-based control programs. The strong heterogeneities in the distribution of ecological and sociodemographic factors associated with house infestation may be used for risk stratification and targeted interventions. Large households residing in small-sized, precarious houses, with few or no livestock or poultry and lower educational levels, appear to be especially vulnerable for Chagas and other infectious diseases. These households and the affected communities may benefit from targeted disease prevention activities channeled through a more vigorous, adequately staffed, primary healthcare system deployed in the affected rural areas. | Title: Ecological and Sociodemographic Determinants of House Infestation by Triatoma infestans in Indigenous Communities of the Argentine Chaco Summary: Indigenous peoples are especially affected by Chagas and other neglected tropical diseases. One of the most numerous ethnic groups in the Gran Chaco region of South America is the Qom (Toba) people. The living conditions of Qom households most likely differ substantially from those of Creoles, and their association with house infestation by triatomine bugs has not been investigated. This is important because the major vector species have adapted to thrive in human sleeping quarters, and in addition to housing construction patterns, other ecological and sociodemographic factors may affect house infestation. We found that Qom households had much higher domestic infestation than Creole ones, in conjunction with more precarious housing, fewer poultry and livestock. The unexpectedly high local residential mobility of Qom households combined with the large fraction of recently-built houses (derived from a rapidly increasing population with a very young age structure during recent decades) may explain why domestic infestation was much lower than expected from the lack of recent insecticide spraying campaigns. Domestic infestation and bug abundance increased with overcrowding and refuge availability, and decreased with household education levels and insecticide use. These results are useful for designing improved interventions and household risk stratification. | 9,524 | 295 | lay_plos | en |
Summarize: The last year brought some difficult moments for Jahqui Sevilla, who escaped from Pulse nightclub after the shooting started. But she was getting better — going to college, working, playing fullback for the Orlando Anarchy women’s tackle football team. Just after midnight Monday she was driving south on State Road 417 when her Mitsubishi drifted into the northbound lanes, hitting another car head-on. She and the other driver, Soraya Matias-Roth of Lakeland, 53, were both killed. “She was getting her life back from when it was stolen,” said Chandice Hunter, the Orlando Anarchy’s team captain. “It just breaks my heart. She’s like a little sister.” Matias-Roth was carrying three passengers, who were injured but are expected to live, said Sgt. Kim Montes, a Florida Highway Patrol spokeswoman. Troopers are still investigating what caused the crash. Sevilla, 20, joined the Anarchy about two years ago, Hunter said. On the field she was driven to succeed and improve, and she didn’t shy away from taking on bigger and tougher opponents. PICTURE GALLERY: Orlando police responded to a mass shooting at Pulse nightclub south of downtown on June 12, 2016. Off the field, she had a goofy sense of humor, Hunter said. She collected things that reminded her of her friends and loved ones, like snap-back hats or small flags or anything with the Batman logo. “She had the best heart and the kindest soul and spirit you could ever imagine,” Hunter said. “She was always looking for the positive in any situation, and she had a smile that could light up anybody’s day, even in the darkest of hours.” Sevilla had just completed her second semester at Valencia College, where she was taking general education courses, college spokeswoman Carol Traynor said. She also worked at a clothing store in the Altamonte Mall, Hunter said, and had talked about becoming a firefighter. Sevilla’s hair, which she wore in short dreads that sprang out of her head, earned her the nickname Piña — Spanish for pineapple. Hunter came back from a trip to Hawaii last week with two new trinkets for Sevilla: a snap-back hat with the word “Aloha” on it, with a pineapple taking the O’s place, and a pineapple key chain. The night of June 11, Sevilla and some of her teammates and friends went to see the Orlando Predators Arena Football League team play, then went to Pulse to dance at Latin Night. As closing time neared, most of the group had left the club. Only Sevilla, her teammate Paula Blanco, and Blanco’s boyfriend Cory Connell were left inside, Hunter wrote in the days after the attack. When the shooting started, Blanco was hit in the forearm. Sevilla kept pressure on the wound, waited until they could safely move and helped guide her outside. Connell was one of the 49 killed in the shooting. On New Year’s Day, Sevilla rode on a float in the Rose Parade in Pasadena, Calif., with two other survivors, Victor Baez Febo and Isaiah Henderson, as well as Pulse owner Barbara Poma. Photos from the parade show Sevilla smiling on the flower-covered float, with a striped gray-and-white scarf around her neck and a rainbow ribbon pinned to her coat. “My heart is so broken with the loss of such a beautiful, pure soul,” Poma wrote on her Facebook page Tuesday morning. “Jahqui Sevilla you are so loved, and I was so blessed to have gotten the chance to be in your life.” In her second-to-last game with the Orlando Anarchy in mid-May, against the Daytona Waverunners, Sevilla saw the ball coming at her and swatted it out of the air instead of catching it, Hunter said. “The ball’s in the air, and out of nowhere she just showed up,” Hunter said. “… We joked on her, but it was actually a fantastic play. It really was.” glotan@orlandosentinel.com or 407-420-5774 0 Pulse nightclub attack survivor dies in Orange County head-on crash ORANGE COUNTY, Fla. - One of two women who died Monday in a head-on crash on the GreeneWay was a survivor of the Pulse nightclub shooting, Orlando City Commissioner Patty Sheehan told Channel 9's Shannon Butler on Tuesday. Sheehan described Jahqui Sevilla as a good person and a private woman who was starting to get her life back together after the June 12, 2016, attack. The 20-year-old Orlando woman was leaving the club last year when Omar Mateen began shooting. Sevilla, her friend, Paula Blanco, and Blanco's boyfriend, Cory Connell, hid behind a couch. In the hours after the attack, Sevilla told Channel 9's Roy Ramos that she carried Connell out of the club after he had been shot, but he died. Read: FHP: 2 women die in head-on crash on GreeneWay in Orange County Heartbreaking: We've just confirmed Jahqui Sevilla, a #Pulse survivor, was killed in a crash Mon morning. Here she is @ Rose Parade. #WFTV pic.twitter.com/E2eIgiOoJO — Cuthbert Langley (@CLangleyWFTV) May 30, 2017 Raw: FHP: 2 women die in head-on crash on GreeneWay in Orange County Troopers said Sevilla was driving south on State Road 417 near University Boulevard at about 12:30 a.m. Monday when she lost control of her Mitsubishi Lancer, crossed the median and crashed head-on into a northbound Dodge Durango driven by Soroya Matias-Roth, 53, of Lakeland. The collision killed both women, investigators said. "I did a lot of crying before you got here. I did a lot of crying last night," said Benijno Sevilla, Jahqui Sevilla's father. "She likes to make people laugh. She has a beautiful smile, beautiful cheeks." Troopers said they're still trying to determine whether alcohol was a factor in the crash. Photos: Fatal crash on SR 417 in Orange County © 2018 Cox Media Group. © 2018 Cox Media Group. Sevilla's father said his daughter's recovery was long after the Pulse shooting, but she made progress. "I was appreciative of the fact we had time. I was glad that she was fortunate enough to survive Pulse. And I appreciate the time after it and I always appreciate hugging and kissing her and telling her how much I love her," he said. Barbara Poma, the nightclub's owner, posted a tribute to Sevilla Tuesday on Facebook. Sevilla and other survivors of the mass shooting rode earlier this year on a tribute float in the 2017 Rose Parade in Pasadena, California. Sevilla was an athlete who played for the Orlando Anarchy women's football team. The crash remains under investigation. New: Jahqui Sevilla's football team releases statement on her death. "Jahqui Sevilla will always be in our hearts..." #WFTV pic.twitter.com/E3Mf9DoK35 — Cuthbert Langley (@CLangleyWFTV) May 30, 2017 © 2018 Cox Media Group. © 2018 Cox Media Group. | Summary: Sad news from Orlando: Jahqui Sevilla, a 20-year-old woman who survived last year's Pulse nightclub massacre, has been killed in a head-on collision less than two weeks before the anniversary. Troopers say Sevilla was driving south on State Road 417 around 12:30am Monday when her Mitsubushi Lancer crossed the median and collided and hit 53-year-old Soraya Matias-Roth's Dodge Durango, WFTV reports. Both women were killed. Sevilla, who played for the Orlando Anarchy tackle football team, was at the nightclub with teammate Paula Blanco and Blanco's boyfriend, Cory Connell, during the June 12 attack. Connell was one of the 49 people killed. Blanco was shot in the forearm and Sevilla helped her escape the building. Friends and family members say Sevilla had some tough times after the mass shooting but was attending college and getting her life back together. "She was getting her life back from when it was stolen," Orlando Anarchy Captain Chandice Hunter tells the Orlando Sentinel. "It just breaks my heart. She's like a little sister." In Pasadena's Rose Parade on New Year's Day, Sevilla rode on a flower-covered float with two other Pulse survivors and nightclub owner Barbara Poma, who posted a photo of herself and Sevilla on Facebook Tuesday. "My heart is so broken with the loss of such a beautiful, pure soul," she wrote. "Jahqui Sevilla you are so loved, and I was so blessed to have gotten the chance to be in your life." | 1,674 | 352 | multi_news | en |
Summarize: This appln claims the benefit of Provisional No. 60/081,352 filed Apr. 10, 1998. FIELD OF THE INVENTION My invention relates to a toy, and more generally, to ornamental objects which may be crushed or otherwise deformed without damage, and which are capable of returning to their original shape after the deforming forces are removed from the object. BACKGROUND A wide variety of children's toys and various ornamental objects are made of rubber compounds which are formulated for resiliency, elasticity, strength, and other important properties. I am aware of various attempts in which an effort has been made to provide an improved toy which has retarded resiliency. Such designs are largely characterized by the use of rubber compounding formulations which result in designs that have a “slow motion” return to the undistorted shape. One such design is shown in U.S. Pat. No. 2,830,402, issued Apr. 15, 1958, to J. A. Jones for ORNAMENTAL TOYS POSSESSING RETARDED RESILIENCY. In one embodiment, his invention provides a solid elongated body of substantial thickness that is made of a resilient material of essentially low molecular weight polyvinyl chloride, and a resiliency retarding ingredient so that the return to the original shape, after deformation, will be relatively slow. OBJECTS, ADVANTAGES, AND NOVEL FEATURES I have now invented, and disclose herein, a novel, design for flexible, resilient foam animals which may be packaged in the form of puzzle parts, and which can be assembled into characters or animals having interchangeable, moveable limbs. When animals are provided, the foam objects are adapted to being interchangeably assembled, and to having limbs articulated about pivot assembly points. My resilient foam objects are simple, lightweight, relatively inexpensive and easy to manufacture, and otherwise superior to those designs heretofore used or proposed, in so far as I am aware. From the foregoing, it will be apparent to the reader that one important and primary object of the present invention resides in the provision of a novel foam toy susceptible of being manually assembled. Other important but more specific objects of the invention reside in the provision of a novel foam toy as described herein which: can be manufactured in a simple, straightforward manner with commonly available manufacturing procedures such as water jet cutting techniques, or with die stamping techniques from commonly available foam materials; in conjunction with the preceding object, have the advantage that they can be easily left in position in the material from which they are formed, in order to provide a “puzzle” having insert shapes corresponding to various body portions for the selected object; which in a relatively inexpensive manner can be provided in a variety of shapes and sizes to produce animals or other objects of any imaginable shape, size, or color. Other important objects, features, and additional advantages of my invention will become apparent to the reader from the foregoing and the appended claims and as the ensuing detailed description and discussion proceeds in conjunction with the accompanying drawing. SUMMARY OF THE INVENTION I have now invented and disclose herein a novel puzzle which can be removed from the puzzle form and converted into a three dimensional object such as a toy foam animal. Such a foam animal preferably includes interchangeable body parts, and more preferably includes articulating appendages which are interchangeably attachable to the body at pivot structures that are affixed to, or which extend thru, the body portions of the foam animal. In one embodiment, pivot structures are formed by resilient foam pivot pins having deformable retaining caps at opposing ends thereof, which, after attachment of articulating limbs, serve to retain the limbs. In another embodiment, the pivot pins act against hex-socket shaped apertures (defined by wall edge portions) that are located on each limb. The novel foam animal provides a simple, interesting, attention focusing toy for children. Various designs can be used to increase the variety of shapes and sizes of the animals, and the toys can be made in the shape and size appropriate to suggest any desired species, whether it be dinosaurs, turtles, farm animals, pets, reptiles or any other selected design. This design provides a most interesting novel structure for assembling toy objects, and especially toy animals, compared to previous designs known to me. BRIEF DESCRIPTION OF DRAWING FIG. 1 is a perspective view of a foam animal, prepared according to the present invention, shown with all parts located in “puzzle” form in a form block from which the object is formed, and indicating in broken lines the removal of an integrally formed animal body section from the foam base. FIG. 2 is a perspective view which illustrates one method of assembly of a foam animal, where first and second body portions are assembled at an interlocking joint, and where front and rear pairs of flexible legs are attached on either side of a fixed type pin which extends through, and protrudes transversly from, the first and second body portions, respectively. FIG. 3 is a side view of the foam animal, similar to that first depicted in FIG. 2, with now illustrating a one-piece body for the animal, and also illustrating front and rear limb pairs fixedly attached at hip and shoulder locations. FIG. 4 is a side view a foam animal similar to that first depicted in FIG. 3 above, but now showing a one-piece body for the animal with front and rear pairs of articulating limbs that are attached to round pivot dowels that are inserted in the body at hip and shoulder locations. FIG. 5 is a partial cross-sectional view of an animal body at a joint location, taken through a pivot dowel, showing the flexible integral end cap on each end of the pivot dowel which releasably secures the limbs to the pivot. FIG. 6 is a an exploded perspective view which illustrates one method of assembly of a foam animal, where first and second body portions are assembled at a substantially horizontally oriented interlocking joint, and where front and rear pairs of flexible legs are attached on either side of an articulating pivot pin which extends through, and protrudes transversly from, the first and second body portions, respectively. FIG. 7 is side view of the assembled foam animal just depicted in FIG. 6, now showing the animal with all parts fully assembled, and indicating in broken lines the alternate position, after movement by pivoting, of the front and rear legs. FIG. 8A is yet another embodiment for a foam animal cow, shown in the unassembled puzzle form, where parts are provided in multiple colors. FIG. 8B is the embodiment just illustrated in FIG. 8A above, now showing a perspective view of a fully assembled foam cow. FIG. 8C again illustrates the embodiment first shown in FIGS. 8A and 8B, now showing a side view of a fully assembled foam cow, more clearly illustrating the provision of various color foam parts. FIG. 8D is yet another view of the foam animal cow in the unassembled puzzle form. FIG. 9A is yet another embodiment for a foam animal dog, shown in the unassembled puzzle form, where parts are provided in multiple colors. FIG. 9B is the embodiment just illustrated in FIG. 9A above, now showing a perspective view of a fully assembled foam dog. FIG. 9C again illustrates the embodiment first shown in FIGS. 9A and 9B, now showing a side view of a fully assembled foam dog, more clearly illustrating the provision of various color foam parts. FIG. 9D is yet another view of the foam animal dog in the unassembled puzzle form. FIG. 10A is yet another embodiment for a foam frog, shown in the unassembled puzzle form, where parts are provided in multiple colors. FIG. 10B is the embodiment just illustrated in FIG. 10A above, now showing a perspective view of a fully assembled foam frog. FIG. 10C again illustrates the embodiment first shown in FIGS. 10A and 10B now showing a side view of a fully assembled foam frog, more clearly illustrating the provision of various color foam parts. FIG. 10D is yet another view of the foam animal frog in the unassembled puzzle form. FIG. 11 again illustrates another embodiment, similar to that shown in FIGS. 8D, 9 D, and 10 D, now showing an elephant in the unassembled puzzle form. FIG. 12 illustrates the embodiment first shown in FIG. 11, now illustrating the side view of a fully assembled foam elephant, also illustrating the use of hexagonal or hex-socket shaped cutouts in leg members to allow adjustable positioning of leg members. FIG. 13 illustrates yet another embodiment, similar to that shown in FIGS. 8D, 9 D, 10 D, and 11, now showing a gorilla in the unassembled puzzle form. FIG. 14 illustrates the embodiment first shown in FIG. 13, now illustrating the side view of a fully assembled foam gorilla, and also illustrating the use of hexagonal or hex-socket shaped cutouts in leg members to allow adjustable positioning of leg members. FIG. 15 shows a universal adaptor, designed for joining any selected pair of first parts (which have male dove tail joints) together. FIG. 16 shows the use of the universal adaptor (just illustrated in FIG. 15 above) for joining a pair of gorilla head portions together to form a “double gorilla” foam animal toy. FIG. 17 shows the use of extended shoulder pins and hip pins to assemble foam animals into multiple animal groups; here, the length of extended shoulder and hip pins allows a triple dinosaur group to be assembled on a pair of common hip and shoulder pins. FIGS. 18A, 18 D, 18 C, 18 D, 18 E, 18 F, 18 G, 18 H, and 18 I represent alternate colors for various parts of the foam toys of the present invention. DESCRIPTION Attention is directed to FIG. 1 of the drawing, where a resilient foam puzzle block 10 is shown. Block 10, nominally of about one-half inch in thickness T, has a plurality of parts removably located therein. As shown, such parts include an elongated, flexible animal body 12, a pair of flexible animal arms 14 and 16, a pair of flexible animal legs 18 and 20, a fixed type shoulder pin 22, and a fixed type hip pin 24. Assembly of the parts into a foam animal is started by detaching the body 12 from block 10 as shown in hidden lines in FIG. 1 as body 12 ′. Then, the fixed type hip pin 24 is detached from block 10 and inserted in the body 12 at complementary sized hip aperture H, in a manner similar to that indicated in FIG. 2. Next, the fixed type shoulder pin 22 is removed from block 10 and inserted into body 12 at complementary sized shoulder aperture S. Finally, flexible animal legs 18 and 20 are attached to hip pin 24 on laterally opposing sides of body 12. Similarly, flexible animal arms 14 and 16 are attached to shoulder pin 22 on laterally opposing sides of body 12. Where fixed type hip pins 24 and fixed type shoulder pins 22 are utilized, I prefer to utilize a parallepiped type shape for each of pins 22 and 24. More preferably, an elongated, rectangular shape is utilized for such pins. Regardless, it is important that the hip socket aperture H and the shoulder socket aperture S in body 12 are of complementary size and shape to the shoulder pin 22 and the hip pin 24, respectively, Likewise, apertures 14 S and 16 S are provided in arms 14 and 16, respectively, and apertures 18 H and 20 H are provided in legs 18 and 20, respectively, each of size and shape to accommodate the respective shoulder pin 22 or hip pin 24. One embodiment of a final, fully assembled animal 30 is shown in FIG. 3, where a fully assembled dinosaur is provided. Kids often find it fun to “mix-and-match” animal body portions, and one structure which lends itself to such practices is illustrated in FIG. 2. A first body portion 32 and a second body portion 34 are provided. An interlocking joint 36 is provided between first body portion 32 and second body portion 34, preferably utilizing a “dove-tail” type of interlocking pattern, where an outwardly expanding wedge shaped tail 38 is provided at the rear 40 of first body portion 32, and where a complementary outwardly expanding receiving aperture 42 is provided at the front portion 44 of the second body portion 34. However, any convenient detachably interlocking joint would be suitable for interlocking the first body portion 32 and the second body portion 34. To increase enjoyment and “action” potential in interactive use of my resilient foam animal designs, I have found it desirable to provide a resilient foam animal 50 which has pivotable front limbs 52 and 54, and pivotable rear limbs 56 and 58, as is shown in FIGS. 4 and 5. In such a design, it can seen that, for example, a rear limb 58 could be pivoted by an angle alpha (α) to the position indicated as 58 ′. Likewise, front limbs 52 and 54 could be pivoted by an angle beta (β), such as the angle shown between front limb 52 and front limb 54. Preferably, there is no limit on either angle alpha (α) or on angle beta (β), and either of front limbs 52 and 54, and either of rear limbs 56 and 58, can be rotated a full three hundred sixty (360) degrees. As better understood from study of FIG. 5, for securing front limbs 52 and 54 or for securing rear limbs 56 and 58 to the body 60, I have found it advantageous to use round foam dowels with integral caps as hip pins 66, as well as for the similar shoulder pins 64. In FIG. 5, an integrally capped round dowel pin, such as hip pin 66, is illustrated in cross-section. Both of the integrally provided caps 70 and 71 extend radially outward from the cylindrical surface 72 of pin 66 by a small pre-selected distance L, so that an inward surface 74 of cap 70 or 71 provides a retaining force against the outer surface face F of a selected limb, whether it be rear limbs 56 and 58 as shown in FIG. 5, or the front limbs 52 and 54. Turning now to FIG. 6, a foam turtle 80 with a two-piece body is provided. A first body portion 82 and a second body or shell portion 84 are interlocked at a substantially horizontally oriented joint, using a dove-tail wedge 86 and a complementary wedge shaped aperture 88, similar that described in FIG. 2 above. A shoulder pivot pin 90 is placed through shoulder socket aperture 92 (defined by wall 93 ), and a first pair of limbs 94 and 96 are attached thereto, and retained thereon, in the manner depicted in FIG. 5 and just described above. Similarly, a hip pivot pin 98 is placed through hip socket aperture 100 (defined by wall 101 ), and second pair of limbs 102 and 104 are attached thereto, and retained thereon in the manner depicted in FIG. 5. Motion is allowed in the front limbs 94 and 96, and in the rear limbs 102 and 104, as indicated in FIG. 7. In FIG. 7, a repositioned front limb 94 is shown in broken lines displaced by an angle epsilon (ε) to a position 94 ′, and where a repositioned rear limb 102 is shown in broken lines displaced by an angle delta (Δ) to a position 102 ′. In each of the limbs 94, 96, 102, and 104, joint apertures J are provided for attachment of the relevant shoulder or hip pivot pins 90 and 98, respectively. Also, in this embodiment, flat foot portions 108 are provided for each of limbs. Additionally, to provide a surface pattern for shell 110, interlocking shell portions 112 and 114 are provided to interfittingly lock into the second body or shell portion 84. Turning now to FIGS. 8A, 8 B, 8 C, and 8 D, yet another embodiment of my invention is depicted. Here, a foam cow is 120 is shown in unassembled form in a foam block puzzle 122, in FIGS. 8A and 8D. In FIG. 8B, a perspective view of an assembled foam cow 120 is provided. In FIG. 8C, a side view of a fully assembled cow 120 is provided, and this figure is shaded for color of one preferred version of the foam cow 120. As is evident in FIG. 8D, front legs 124 and 126, and rear legs 128 and 130 are provided. A head portion 132 is provided, and a rear body portion 134 is provided. Front legs 124 and 126 are affixed to head portion 132 via shoulder pin 136. Rear legs 128 and 130 are affixed to the rear body portion 134 via hip pin 138, in the manner described above. Subscripts “S” and “H” are used in conjunction with numbering for front and rear legs to denote apertures for shoulder and hip joints, respectively, in the applicable front and rear legs. In addition, color patches 140 a, 140 b, and 140 c are provided to enhance the visual appearance of the foam cow 120. FIGS. 9A, 9 B, 9 C, and 9 D, depict yet another embodiment of my invention, similar in concept to that just described in FIGS. 8A, 8 B, 8 C, and 8 D. Now, a foam dog is 150 is shown in unassembled form in a foam block puzzle 152, in FIGS. 9A and 9D. In FIG. 9B, a perspective view of dog 150 is provided. In FIG. 9C, a side view of a fully assembled dog is provided, and this figure is shaded for color of one preferred version of the foam dog. As is evident in FIG. 9D, front legs 154 and 156, and rear legs 158 and 160 are provided. A head portion 162 is provided, and a rear body portion 164 is provided. Front legs 154 and 156 are affixed to head portion 162 via shoulder pin 166. Rear legs 158 and 160 are affixed to the rear body portion 164 via hip pin 168, in the manner described above. Subscripts “S” and “H” are used in conjunction with numbering for front and rear legs to denote apertures for shoulder and hip joints, respectively, in the applicable front and rear legs. FIGS. 10A, 10 B, 10 C, and 10 D, depict yet another embodiment of my invention, similar in concept to that just described in FIGS. 9A, 9 B, 9 C, and 9 D. Now, a foam frog is 170 is shown in unassembled form in a foam block puzzle 172, in FIGS. 10A and 10D. In FIG. 10B, a perspective view of frog 170 is provided. In FIG. 10C, a side view of a fully assembled dog is provided, and this figure is shaded for color of one preferred version of the foam dog. As is evident in FIG. 10D, front legs 174 and 176, and rear legs 178 and 180 are provided. A head portion 182 is provided, and a rear body portion 184 is provided. Front legs 174 and 176 are affixed to head portion 182 via shoulder pin 186. Rear legs 178 and 180 are affixed to the rear body portion 184 via hip pin 188, in the manner described above. Subscripts “S” and “H” are used in conjunction with numbering for front and rear legs to denote apertures for shoulder and hip joints, respectively, in the applicable front and rear legs. In addition, color patches 190 a, and 190 b are provided to enhance the visual appearance of the foam frog 170. As illustrated, the animals shown in FIGS. 8C, 9 C, 10 C and related figures utilize fixed limbs similar to the structure first set forth in FIGS. 1 and 2 above, however, it should be understood that these foam animal designs can also be fabricated using the articulating limb structures, either as earlier described with reference to FIGS. 4 and 5, or as described below with reference to the use of differential friction cutout joint sockets to allow repositionable articulation of limbs. Turning now to FIGS. 11-14, two additional embodiments are provided to illustrate the use of a differential friction cutout joint socket that allows the repositionable articulation of a rotatable part such as a limb. First, in FIG. 11, a foam elephant 200 is shown in unassembled form in a foam block puzzle 202. In FIG. 12, a side view of a fully assembled foam elephant 200 is provided. As is evident in FIG. 12, front legs 204 and 206, and rear legs 208 and 210 are provided. A head portion 212 is provided, and a rear body portion 214 is provided. Front legs 204 and 206 are affixed to head portion 212 via shoulder pin 216. Rear legs 208 and 210 are affixed to the rear body portion 214 via hip pin 218. Subscripts “SAF” and “HAF” are used in conjunction with numbering for front and rear legs to denote the unique articulating friction apertures for shoulder and hip joints, respectively, in the applicable front and rear legs. Note that the front leg aperture 204 SAF is defined by an interior, hex-socket shaped wall 220 against which the rectangular end 222 of shoulder pin 216 frictionally engages. Both the wall 220 and the shoulder pin 216 are sufficiently deformable that the front leg 204 may be turned, and then repositioned at a new location corresponding to new, preferably opposing wall portions 220 x-1 and 220 x-2 of the interior socket-shaped wall of the front leg aperture 204 SAF. Similarly, interior, hex-socket shaped wall 224 is provided in the other front leg 206, and hex-socket shaped walls 226 and 228 are provided in the rear legs 208 and 210, respectively. Although the elephant 200 shown in FIGS. 11 and 12 is not shaded for color, any of the parts may be manufactured in any desired and available color foam, as indicated by the reference to FIGS. 18A through 18I, which represent alternate colors for the body of the foam elephant 200, as shown in FIG. 11. Moreover, any one of the parts of the embodiments illustrated herein, or alternate embodiments built in accord with the teachings hereof, or the legal equivalents thereof, may be manufactured in any of the alternate colors selected from those illustrated in FIGS. 18A through 18I. The differential friction cutout joint socket that allows the repositionable articulation of a rotatable part such as a limb is further depicted in FIGS. 13 and 14, where a foam gorilla 240 is depicted. In FIG. 13, the foam gorilla 240 is shown in unassembled form in a foam block puzzle 242. In FIG. 14, a side view of a fully assembled foam gorilla 240 is provided. As is evident in FIG. 13, front legs 244 and 246, and rear legs 248 and 250 are provided. A head portion 252 is provided, and a rear body portion 254 is provided. Front legs 244 and 246 are affixed to head portion 252 via shoulder pin 256. Rear legs 248 and 250 are affixed to the rear body portion 254 via hip pin 258. Subscripts “SAF” and “HAF” are used in conjunction with numbering for front and rear legs to denote the unique articulating friction apertures for shoulder and hip joints, respectively, in the applicable front and rear legs. Note that the front leg aperture 244 SAF is defined by an interior, hex-socket shaped wall 260 against which the rectangular end 262 of shoulder pin 256 frictionally engages. Both the wall 260 and the shoulder pin 256 are sufficiently deformable that the front leg 244 may be turned, and then repositioned at a new location corresponding to new, preferably opposing wall portions 260 x-1 and 260 x-2 of the interior socket-shaped wall of the front leg aperture 244 SAF. To turn front leg 244 by an angle A, the rectangular end 266 of shoulder pin 256 is brought to bear against interior wall portion 260 x-1 of front leg 244. Likewise, to turn front leg 244 by an angle B, the rectangular end 266 of shoulder pin 256 is brought to bear against interior wall portion 260 x-2 of of front leg 244. Similarly, interior, hex-socket shaped wall 264 is provided in the other front leg 246, and hex-socket shaped walls 266 and 268 are provided in the rear legs 248 and 250, respectively. As now thoroughly illustrated, appendages such as legs 244 and 248 can be articulated about a shoulder pin 256, for easy manipulation. Similar structures are provided in other appendages. Turning now to FIG. 15, a unique mating joint 300 is illustrated. The joint is preferably generally H-shaped, and configured with angular wing portions to lockingly accept, on each of opposing sides 310 and 312, a wedged shaped or dove tail element DT. Thus, a first element (here gorilla head portion 252 ) which includes a dove tail DT, can be joined by the use of mating joint 300 with a second element (here a second gorilla head portion 252 ) which includes a dove tail DT. Generally, this type of interlocking joint has been already described above. Finally, it is interesting to children to join a plurality of “n” foam animals A in a series A 1, A (n−1), through A n. As shown in FIG. 17, n=3, and extended shoulder pins 408 and extended hip pins 410 are provided to extend through the preselected number n of animals A, in order to hold the plurality of animals A together, as well as allow attachment of front legs thereto, in either a fixed or an articulating manner. When fully assembled, as illustrated, a triple dinosaur 420 results. I prefer to build the foam animals or other resilient foam objects in a high strength foam, such as an EVA (ethylene vinyl acetate) or neoprene type foam. However, a variety of suitable materials may be selected for particular designs, without departing from the spirit and details of my invention. It is a fundamental and important quality of my invention that in the preferred embodiment, the foam animals are composed of material which provides a resilient, springing return to the original shape and size after being crushed by hand. Also, the combination of the materials of construction and the long, flexible limb design lends itself to allowing the animal to “jump” when the limbs, particularly rear limbs 56 and 58, for example, are deformed against a flat surface and then released. It is to be appreciated that the novel puzzles, and the resilient foam animals provided by the present invention are a novel and interesting development in the toy manufacturing industry. My novel foam products are relatively simple, and without much cost and complexity, a unique educational toy is provided. It is thus clear from the heretofore provided description that my novel resilient foam toys, as described and disclosed herein, are an appreciable improvement in the state of the art of resilient toys. Although only a few exemplary embodiments of this invention have been described in detail, it will be readily apparent to those skilled in the art that the my novel resilient foam toy animals, or other foam toy objects, may be modified from those embodiments provided without materially departing from the novel teachings and advantages provided by this invention, and may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. Therefore, the embodiments presented herein are to be considered in all respects as illustrative and not restrictive. As such, the claims are intended to cover the structures described herein, and not only structural equivalents thereof, but also equivalent structures. Thus, the scope of the invention is intended to include variations from the embodiments provided which are nevertheless described by the broad meaning and range properly afforded to the language set forth herein, or to the equivalents thereof. | Summary: Deformable foam toys and puzzles. Novel resilient foam puzzles can be removed from puzzle form and converted into a three dimensional objects, such as toy foam animals. The objects preferably include interchangeable parts, and more preferably include articulating appendages which are interchangeably attachable to a body portion at pivot structures that are affixed to, or which extend thru, the body portions. Preferably, pivot structures are formed by resilient foam pivot pins having deformable retaining caps at opposing ends thereof, which, in the case of foam animals, serve to retain articulating limbs. Alternately, a hex-shaped socket aperture defined by a wall edge portion is provided as a joint socket in one or more of the limbs, and a parallepiped, and preferably an elongated rectangular shoulder pin or hip pin is provided for displaceable frictional engagement with the hex shaped socket to articulate the limbs to desired positions. In animal form, such foam articles provide an interesting, attention focusing toy for children. Various designs can be used to increase the variety of shapes, sizes, and colors. The toys can be made in the shape and size appropriate to suggest any desired objects or animal species. The design provides an interesting novel structure for assembling toy animals. | 7,099 | 276 | big_patent | en |
Write a title and summarize: It is well established that RNA viruses exhibit higher rates of spontaneous mutation than DNA viruses and microorganisms. However, their mutation rates vary amply, from 10−6 to 10−4 substitutions per nucleotide per round of copying (s/n/r) and the causes of this variability remain poorly understood. In addition to differences in intrinsic fidelity or error correction capability, viral mutation rates may be dependent on host factors. Here, we assessed the effect of the cellular environment on the rate of spontaneous mutation of the vesicular stomatitis virus (VSV), which has a broad host range and cell tropism. Luria-Delbrück fluctuation tests and sequencing showed that VSV mutated similarly in baby hamster kidney, murine embryonic fibroblasts, colon cancer, and neuroblastoma cells (approx. 10−5 s/n/r). Cell immortalization through p53 inactivation and oxygen levels (1–21%) did not have a significant impact on viral replication fidelity. This shows that previously published mutation rates can be considered reliable despite being based on a narrow and artificial set of laboratory conditions. Interestingly, we also found that VSV mutated approximately four times more slowly in various insect cells compared with mammalian cells. This may contribute to explaining the relatively slow evolution of VSV and other arthropod-borne viruses in nature. RNA viruses show extremely high genetic variability and rapid evolution, ultimately due to their elevated rates of spontaneous mutation, which range from 10−6 to 10−4 substitutions per nucleotide per round of copying (s/n/r). However, mutation rate estimates vary considerably, even for the same virus [1], [2]. Since viral mutation rates have implications for pathogenesis [3], [4], vaccine development [5], [6] antiviral therapy [7], [8], and epidemiological disease management [9], [10], it is important to have accurate data and a clear understanding of the factors determining these rates. As a case in point, the risk of cross-species transmission is determined, in addition to the ecology of virus-host interactions, by the input of new adaptive mutations in the viral population [11], and a recent phylogenetic analysis of rabies virus isolates suggested that the waiting time required for host jumps depends on the number of positively selected mutations involved in cross-species transmission [12]. In RNA viruses, mutation rates are determined by the intrinsic base selection specificity of the viral polymerase [13]–[16], the presence/absence of proofreading mechanisms such as 3′exonuclease activity [17]–[19], or the mode of replication [20], [21]. However, in addition to these virus-encoded factors, viral mutation rates can be host-dependent. For instance, it has been suggested that the replicase of cucumber mosaic virus exhibits different fidelity in pepper and tobacco plants [22], [23]. In retroviruses, replication fidelity may be affected by intra-cellular dNTP imbalance and total concentration, which vary among cell types [24]–[26], although a recent study revealed no differences in the HIV-1 mutation rate in various cell types including T lymphoblast, glioblastoma and human embryonic kidney cells [27]. Also, the expression of host genes may influence the viral mutation rate as is the case of APOBEC3 cytidine deaminases, which can edit the HIV-1 cDNA and produce G-to-A hypermutations [28]–[30]. A similar role was postulated for the cellular RNA-dependent adenosine deaminase (ADAR) which could lead to A-to-G hypermutation in several RNA viruses, including rhabdoviruses [31], paramyxoviruses [32], and retroviruses [33]–[35]. Finally, cell metabolism may also have an impact in viral mutation rates, since it has been shown that ethanol-derived reactive oxygen species (ROS) can damage the RNA of hepatitis C virus, whereas other compounds such as glutathione and iron chelators were found to have the opposite effect [36]. Vesicular stomatitis virus (VSV) is a non-segmented negative-stranded RNA virus belonging to the family Rhabdoviridae with an extremely wide host tropism. The virion attaches to phosphadtidyl serine or other ubiquitous cell surface receptors and can productively infect most mammalian cells [37]. In nature, VSV infects a very large number of mammal species including livestock (cattle, horse, swine, goats, etc.) and wild animals (rodents, bear, lynx, bats, etc.), and also infects insects (sandflies, blackflies, mosquitoes, etc.) [38], [39], which act as transmission vectors [40]–[42]. Therefore, VSV replicates in widely different cellular environments, but the impact of this heterogeneity on the viral mutation rate is unknown. Actually, nearly all mutation rate estimates for animal viruses have been obtained in standard laboratory cell lines, which are usually immortalized or cancerous and thus show aberrant metabolic/mitotic rates and gene expression patterns. For VSV, most studies are conducted using hamster kidney cells, despite the fact that the brain is the main target organ of rhabdoviruses. Furthermore, all viral mutation rate studies have been conducted under atmospheric oxygen levels but these are substantially higher than those found in most tissues [43], and the impact of this type of environmental stress in the estimates is unknown. Here, we measured the mutation rate of VSV in primary and tumoral cell types including murine fibroblasts of various origins and neural cells, and under different oxygen levels, as well as insect cells. We found that the VSV mutation rate was relatively constant in all mammalian cells tested. However, VSV mutated four times more slowly in insect cells than in mammalian cells, a finding that may have implications for our understanding of arboviral evolution. We measured the mutation rate of VSV by the Luria-Delbrück fluctuation test, a standard estimation method [44] that has been used previously in several viruses including poliovirus [45], vesicular stomatitis virus [46], influenza A virus [47], measles virus [48], turnip mosaic virus [49], and bacteriophages φ6 [20] and Qβ [50]. To score mutants, we used a monoclonal antibody against the envelope glycoprotein G and determined the probability of appearance of monoclonal antibody resistance (MAR) mutants in independent cultures (null-class method). First, we performed six independent tests in baby hamster kidney cells (BHK-21), for which we had previous results [46]. This gave an average mutation rate to the MAR phenotype of m = (1. 64±0. 27) ×10−5 per round of copying (Table 1). This rate can be converted to per-nucleotide units as, where T is the set of observable mutations leading to the phenotype (mutation target) and three stands for the number of possible nucleotide substitutions per site [2]. Sequencing of the glycoprotein G gene from 15 MAR plaques allowed us to identify four different nucleotide substitutions, which led to amino acid changes D257N, D259A, D259N, and S273T, whereas previous work reported the same substitutions at position 259 of the G glycoprotein in addition D257G, D257V, D257Y and A263E [51]. Taking T = 8, the estimated mutation rate is μ = 6. 15×10−6 substitutions per nucleotide per round of copying (s/n/r). To verify the reliability of the above estimate, we used a molecular clone sequencing approach. This allowed us to score mutations more directly than in fluctuation tests and to analyze a wider genome region, although the interpretation of the data is complicated by the fact that the observed mutation frequency is dependent on selection, the number of generations elapsed, etc. BHK-21 cells were infected with a single infectious particle (i. e. plaque forming unit, pfu) by limiting dilution, and the resulting viral bursts (1. 55×107 final pfu on average) were used for RNA purification, RT-PCR, molecular cloning, and sequencing of three genome regions mapping to genes P, G, and L. We observed four single-nucleotide substitutions in 77500 bases in total, giving a mutation frequency of f = 5. 16×10−5 (Table 2). For a per-cell burst size of B = 1250 [46], the number of infection cycles (i. e. viral generations) elapsed should be. Therefore, the per-generation increase in mutation frequency was. To account for the effect of selection, we used the previously characterized distribution of mutational fitness effects (see Methods). Based on this, the expected fraction of observable mutations after 2. 3 generations was 53% and, thus, the estimated per-cell mutation rate is. The exact number of round of copying per cell is unknown but a previous work suggested rC = 5. 8 rounds/cell, implying that μ = 7. 30×10−6 s/n/r. This estimate is fully consistent with the results provided by the Luria-Delbrück fluctuation test. Subsequent experiments were done using fluctuation tests only because they provided a faster and simpler approach. Previous mutation rate studies with VSV have been conducted in BHK-21 cells only [2], [46], [52]. However, these are immortalized/tumoral cells, as opposed to those typically encountered bythe virus in vivo. Furthermore, VSV has a tropism for neural cells, and kidney fibroblasts are not a natural target of the virus. Toaddress the potential effect of immortalization on the viral mutation rate, we performed fluctuation tests in primary mouse embryonic fibroblasts (MEFs) and isogenic, p53 knock-out, MEFs. The average rate was similar in normal (m = 1. 27×10−5) and p53knock-out MEFs (m = 0. 82×10−5), revealing no significant effectof cellular immortalization (Figure 1; t-test: P = 0. 232, n = 6). However, many cell lines are tumoral and show other genetic andmetabolic alterations in addition to p53 inactivation. To check the potential effects of these changes, we performed fluctuation tests in CT26 cells from an undifferentiated grade IV colon adenocarcinoma of a BALB/c mouse [53], but we found no significant differences with primary MEFs (m = 1. 18×10−5; t-test: P = 0. 885, n = 6). Of note, BHK-21 are also tumor-forming cells, and the mutation rate was similar to the rate observed in MEFs or CT26 cells (one-way ANOVA: P = 0. 293, n = 12). This homogeneity in the VSV mutation rate was not an obvious a priori, because metabolic and mitotic activity should alter the availability of NTPs [54] and hence could impact RNA replication fidelity, although VSV replicates in the cytoplasm and may not be strongly affected by these alterations. This result has implications for the field of oncolytic virotherapy [55], since it is critical to assess the genetic stability of these therapeutic viruses during large-scale manufacturing and clinical use. In particular, CT26 cells have been used in mice as a model for testing the oncolytic activity of VSV [56]. Also, the above results suggest that VSV replicates with similar fidelity in different cell types, but we sought to test whether this would also hold for neural cells. We therefore performed fluctuation tests in Neuro-2a cells from a mouse neuroblastoma [57]. Again, we found that the average mutation rate did not significantly differ from the rate obtained in BHK-21 cells (m = 1. 06×10−5; t-test: P = 0. 461, n = 9). Finally, to test for other potential effects of cell physiology, we also varied oxygen levels. The VSV mutation rate in BHK-21 cells cultured under hypoxic conditions (1% oxygen) was slightly higher but not significantly different to the rate obtained under standard conditions (m = 2. 71×10−5; t-test: P = 0. 122, n = 9). Oxidative stress should lead to the release of ROS, which have been previously shown to be mutagenic for hepatitis C virus [36]. However, VSV does not appear to be sensitive to oxidation levels. This might be related to the fact that the nucleocapsid of mononegavirales forms a tunnel-like structure which wraps the viral genomic RNA and remains assembled during the entire infection cycle [58], [59], effectively isolating the viral RNA [60]. Since VSV alternates between mammalian and insect hosts in nature, we sought to measure the viral mutation rate in insect cells (Figure 1). In S2 cells from D. melanogaster embryos, the average estimate from three independent fluctuation tests was m = 4. 08×10−6, representing a fourfold decrease compared with BHK-21 (t-test: P = 0. 009, n = 9). To further investigate this, we selected two additional insect cells lines: Sf21 ovarian cells from the moth Spodoptera frugiperda, and C6/36 from Aedes albopictus mosquito larvae. Also, since insect cells were infected at 28°C and mammalian cells at 37°C, we performed four additional tests in BHK-21 at 28°C. We used estimates obtained in mammalian (BHK-21, BHK-21 at 28°C, MEF, MEF p53−/−, CT26, and Neuro-2a) and insect cells (S2, Sf-21, and C6/36) to jointly test for the effects of host type and temperature (fixed factors) in a two-way ANOVA in which the specific cell line was treated as a random factor nested within host type. This confirmed that VSV shows lower mutation rate in insect cells than in mammalian cells (ANOVA: P<0. 001), and also that temperature cannot account for this result because the estimates in BHK-21 were actually higher at 28°C than at 37°C (P = 0. 001). Using log10-transformed data, the estimated effect size of the host type in the above model was 0. 590±0. 205, which implies a 3. 9 fold mutation rate decrease in insect cells. One possible explanation for this difference is that our sensitivity to detect MAR mutants varied between assays performed in mammalian and insect cells. To address this, we first verified that MAR plating efficiency was similar in BHK-21, S2, Sf21, and C6/36 cells using a genetically engineered MAR mutant (D259A). Second, we tested for differences in the mutation target size (T). To do this, we sampled 15 individual MAR plaques from fluctuation tests performed in S2 cells and sequenced the region of the G protein controlling this phenotype. We found the same amino acid replacements as in fluctuation tests performed in BHK-21 cells (D257N, D259N, S273T, see above) except for D259A. However, because the D259 mutant is viable in insect cells [61], failure to detect it was probably due to insufficient sampling depth. We also found substitution A263E, which was reported previously in BHK-21 cells [51]. Therefore, insect S2 and BHK-21 cells shared a similar mutational repertoire and plating efficiency, supporting the consistency of the observed mutation rate difference. Interestingly, VSV [62] and arboviruses in general [63], [64] tend to evolve more slowly than directly transmitted viruses. Our own meta-analysis using 170 previously published evolutionary rates confirmed that, after accounting for phylogenetic relatedness and the timespan of sequence sampling, arboviruses showed a significantly lower evolution rate than directly transmitted viruses (Figure 2; two-way ANOVA: P = 0. 006), the geometric mean rates being 5. 7×10−4 substitutions per site per year (s/s/y) and 1. 3×10−3 s/s/y, respectively. This has been often interpreted in terms of fitness tradeoffs, whereby neutral or beneficial mutations in mammals can be deleterious in insects, and vice versa, thus restricting viral evolution. However, whether arboviruses show similar mutation rates in mammalian and insect cells has not been addressed before, and our results offer a new possible explanation for the relatively slow arboviral evolution. Future experiments with other arboviruses could help elucidate the generality of these findings and, if so, to delineate the mechanisms behind the observed differences in replication fidelity. Viruses were obtained from an infectious cDNA clone by transfecting baby hamster kidney (BHK-21) cells [65], [66], purified by filtration (0. 22 µm), and stored at 70°C in aliquots until use. BHK-21 cells (American Type Culture Collection, ATCC) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 0. 02 mM L-Glutamine, a mix of non-essential amino-acids, 100 µg/mL streptomycin, 60 µg/mL penicillin, and 2 µg/mL fungizone. MEFs and their p53−/− derivatives were obtained from Dr. Carmen Rivas (Centro Nacional de Biotecnología, Madrid) and cultured in the same medium but with 12% FBS. Neuro-2a cells were obtained from Prof. José M. García-Verdugo (Department of Cell Biology, University of Valencia) and cultured in MEM supplemented with 2 mM L-Glutamine, 1 mM sodium pyruvate, 10% FBS, non-essential amino acids and the above antibiotics. CT26 cells (ATCC) were cultured in DMEM with 10% FBS, 2 mM L-glutamine, 10 mM HEPES and antibiotics. All the above cells were incubated at 37°C with 5% C02 and passaged upon confluence. D. melanogaster Schneider (S2) cells were obtained from Dr. Rubén Artero (Department of Genetics, University of Valencia) and cultured in Schneider' s medium supplemented with 10% FBS and antibiotics at 25°C in the absence of C02, and infected at 28°C. Sf21 cells were obtained from Dr. Salvador Herrero (Department of Genetics, University of Valencia) and were cultured in Grace' s insect medium supplemented with 10% FBS and antibiotics at 28°C in the absence of C02. C6/36 cells (ATCC) were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, non-essential amino acids, 1 mM sodium pyruvate and antibiotics at 28°C under 5% C02. Hypoxia was achieved by displacing oxygen with nitrogen, using a Galaxy 170R incubator (Eppendorf). We inoculated 32 identical cultures each containing 104 confluent cells with approximately 300 pfu/well (Ni) and incubated them until approximately 3×104 pfu/well were produced (Nf). After a round of freeze-thawing to release intracellular particles, we used eight cultures for titration and 24 for plating the entire undiluted volume (100 µL) in the presence of a monoclonal antibody against the surface glycoprotein G at a concentration that neutralizes completely the wild-type virus and selects for MAR mutants. The antibody, in the form of a hybridoma supernatant, was added to the plating medium (25% v∶v) to avoid phenotypic masking [52]. Plating assays were done in DMEM gelled with 0. 4% agarose containing 2% FBS. After 24 h, monolayers were fixed with 10% formaldehyde and stained with 2% crystal violet to visualize plaques. Since mutation is a rare event, the number of mutations per culture is expected to follow a Poisson distribution of parameter and therefore the probability of observing no mutants in a culture is, where m is the mutation rate from the wild-type to the MAR phenotype (null-class method). However, if there is incomplete plating, some cultures may contain undetected MAR mutants. If we define z as the plating efficiency (relative to BHK-21 cells), the probability of observing no mutants can be expressed as, where is the probability of k actual mutants in a culture. Using a Poisson distribution of parameter for k, we numerically solved Q0 given P0, Ni, Nf, and z and calculated the mutation rate as. For each cell type tested, the plaque assay for scoring MAR mutants was done in BHK-21 cells for technical feasibility and to control for differences in plating efficiency among cells. However, since plaque assays to score MAR mutants were done without dilution, antiviral cytokines or other compounds released from the cells in which the virus was grown could modify plating efficiency (plaque assays for determining Nf were done at a roughly 1/100 dilution and thus were much less affected by this problem). For instance, BHK-21 cells are at least partially responsive to interferon [67], potentially inhibiting growth of MAR mutants and biasing mutant counts down. To calibrate this effect, we titrated a MAR clone obtained by site-directed mutagenesis (substitution D259A in the surface glycoprotein G) in the presence of undiluted supernatants harvested from cells previously infected with the wild-type virus (Ni≈300 pfu and Nf>≈104 pfu, similar to fluctuation tests), adding monoclonal antibody to the plates to observe MAR plaques only. The wild-type infections were performed under each of the experimental conditions (BHK-21, MEF, MEF p53−/−, CT26, Neuro-2a, BHK-21 with 1% O2, BHK-21 at 28°C, S2, Sf21 and C6/36 cells). Addition of supernatants from BHK-21 cells infected under standard conditions did not alter the titer of the D259 MAR clone, hence the relative plating efficiency was z = 1. The relative plating efficiency for each of the other conditions is shown in the Supporting Information “Text S1” and was based on at least six independent plating assays. To ascertain the number of possible mutations conferring the MAR phenotype, we plated approximately 105 pfu in the presence of antibody, incubated them for 24 h, and pipetted individual plaques. Viral RNA was purified, reverse-transcribed using AccuScript High Fidelity Reverse Transcripatse (Agilent Technologies), and the cDNA was PCR-amplified using Phusion High Fidelity DNA polymerase (New England Biolabs). We used specific primers to amplify and sequence a region of the G protein (genome sites 3361 to 4501 in GenBank accession EF197793) which controls the MAR phenotype [51]. PCR products were sequenced by the Sanger method and analyzed using Staden software. A 96-well plate containing 104 cells per well was inoculated with a limiting dilution of the viral stock such that approximately 10% of wells were infected. Plates were incubated at 37°C for 24 h, inspected under the microscope for cytopathic effects, and freeze-thawed to allow release of intracellular viruses. Viral RNA was purified from the supernatant of each of five positive wells and reverse-transcribed using AccuScript High Fidelity Reverse Transcripatse, and the cDNA was PCR-amplified using Phusion High Fidelity DNA polymerase and specific primers located in the P, G and L genes, as indicated. PCR products were cloned and used for E. coli transformation, and 10 colonies were picked and amplified by colony PCR using Phusion High Fidelity DNA polymerase. PCR products were sequenced by the Sanger method and analyzed using Staden software. To obtain the mutation frequency, the number of observed mutations was divided by the total number of bases sequenced. We used the empirically characterized distribution of mutational fitness effects of random single-nucleotide substitutions in VSV to correct for the effect of selection on mutation frequency and obtain the mutation rate per cell infection. We did so numerically by simulating the combined effects of mutation and selection. The statistical distribution of fitness effects (s) for viable substitutions can be roughly captured using an exponential distribution truncated at (lethality) plus a class of lethals occurring with probability pL: if 0<s<1, if s = 1, and otherwise. In a previous work using the same VSV strain as here, it was estimated that and that [65], [68]. Fitness effects were measured as growth rate ratios,, where r is the exponential growth rate and subscripts i and 0 refer to the mutant and wild-type, respectively [65]. These s-values were transformed to per cell infection units as, where B is the burst size. After simulating fitness effects using the truncated exponential plus lethal distribution and applying the per cell infection transformation, selection was applied by picking individuals for the next cell infection cycle with weighted probability 1−s′, and the process was iterated. This provided an expected mutation frequency f and therefore a relationship between μ and f. Genetic drift was ignored since it should not modify the expected value of f. Also, for simplicity, mutations were assumed to have independent fitness effects (no epistasis) and back mutations were ignored, which seems reasonable in the short-term, because single forward mutations will greatly outnumber secondary and back mutations. Simulations were performed using Wolfram Mathematica and Excel. A graphical representation of this correction can be found in a previous work [2]. In a previous meta-analysis, we collected evolutionary rate estimates that were originally inferred from field isolates using Bayesian analysis of dated sequences after validation of the molecular clock [69]. Here, we used 170 of these estimates, which corresponded to 62 different riboviruses. We sought to compare viruses transmitted directly through respiratory secretions, blood, sexual contact, feces, or animal bites (n = 113) against arboviruses (n = 57). We used a two-way ANOVA in which the following factors were included: transmission mode (fixed), viral family (random) to account for phylogenetic relatedness, and sampling timespan (covariate) to account for the known time-dependency of evolution rate estimates. Since rates ranged several orders of magnitude log-transformed data were used. | Title: Variation in RNA Virus Mutation Rates across Host Cells Summary: RNA viruses show high rates of spontaneous mutation, a feature that profoundly influences viral evolution, disease emergence, the appearance of drug resistances, and vaccine efficacy. However, RNA virus mutation rates vary substantially and the factors determining this variability remain poorly understood. Here, we investigated the effects of host factors on viral replication fidelity by measuring the viral mutation rate in different cell types and under various culturing conditions. To carry out these experiments we chose the vesicular stomatitis virus (VSV), an insect-transmitted mammalian RNA virus with an extremely wide cellular and host tropism. We found that the VSV replication machinery was robust to changes in cellular physiology driven by cell immortalization or shifts in temperature and oxygen levels. In contrast, VSV mutated significantly more slowly in insect cells than in mammalian cells, a finding may help us to understand why arthropod-borne viruses tend to evolve more slowly than directly transmitted viruses in nature. | 6,419 | 226 | lay_plos | en |
Summarize: On Tuesday, less than a week after the death of Playboy founder Hugh Hefner, the news broke that his life will be turned into a biopic starring none other than Jared Leto. The Academy Award-winning actor is in the early stages (so early that there's neither a release date nor a title for it just yet) of making a movie about Hefner’s life with producer Brett Ratner, who’s slated to direct. "Jared is an old friend," Ratner told The Hollywood Reporter. "When he heard I got the rights to Hef's story, he told me, 'I want to play him. I want to understand him.' And I really believe Jared can do it. He's one of the great actors of today." Leto isn’t just an actor: he’s also an avid mountain climber (albeit one who scales the slopes wearing copious amounts of Gucci) and the frontman of the band 30 Seconds to Mars. Word has it he never had the opportunity to meet Hefner in person, but that shouldn’t stop him from turning out a great performance. Hefner lived his life in full view of the public for well over 60 years, and with talk shows, reality shows, and even movie and TV cameos, his mannerisms and personal style should be easy for Leto to capture. And if Ray, The King's Speech, and Braveheart are any indication, this film has a good chance of setting him up to win another Oscar. In the meantime, his next movie, Blade Runner 2049, will premiere on Friday, Oct. 6. Jared Leto has officially signed on to play the late Playboy founder Hugh Hefner in an upcoming biopic, according to the Hollywood Reporter. The announcement comes just a week after Hefner passed away at 91, but the film's reportedly been in the works for the better part of a decade. The Hef biopic is the brainchild of Brett Ratner, the guy who gave us Rush Hour and directed the goofiest X-Men movie ever made. Ratner's been scrambling to tell Hefner's weird-ass life story for years, at one point nabbing Robert Downey Jr. to star before the project went haywire, according to the Reporter. In April, the Big Butt Book–loving director finally got the greenlight to make the movie—and, as he tells it, his "old friend" Leto became the leading contender for the starring role almost immediately. "When [Leto] heard I got the rights to Hef's story, he told me, 'I want to play him. I want to understand him,'" Ratner told the Reporter. "And I really believe Jared can do it. He's one of the great actors of today." Ratner even took Leto down to the Playboy mansion for a premiere of the Amazon docuseries American Playboy: The Hugh Hefner Story, which also doubled as the tycoon's 91st birthday party. Ratner had hoped to introduce Leto to the onetime Curb guest star in person, but apparently Hef wasn't feeling well enough for the visit. Still, Ratner's convinced the actor will handle the role just fine. "There's enough footage on Hef out there that Jared will be able to get as much information as he wants," he told the Reporter. Leto just finished wrapping up Blade Runner 2049, and he's still slated to play Andy Warhol in another upcoming biopic. The Hefner movie is still in early development, and there's no timeline about when it will go into production, but whenever it does, Leto's co-stars better get ready for some gross-ass gifts behind the scenes. In some news that we weren’t hoping to start our day off with, The Hollywood Reporter has revealed that Jared Leto will take on the role of Playboy founder Hugh Hefner, who died just last week at the age of 91, for Brett Ratner’s long-in-development biopic surrounding the late media mogul. The project has been in limbo since 2007 when it was originally set up at Universal Pictures and Imagine Entertainment. However, the film sat on the shelf for a number of years and the rights soon expired, leading to them being purchased by producer Jerry Weintraub for Warner Bros. Advertisement “Jared is an old friend,” Rater, who acquired the rights to the project after Weintraub’s death in 2015, told THR. “When he heard I got the rights to Hef’s story, he told me, ‘I want to play him. I want to understand him.’ And I really believe Jared can do it. He’s one of the great actors of today.” I think we can all agree that this biopic will shy away from being an honest retelling of Hefner’s actual life story and will more so serve as a celebration of his life, to which I say, in the words of Ira Madison III: “Keep it.” The producer of the upcoming Hefner biopic believes the Playboy founder and "consummate optimist" was one of a handful of leaders who made American culture less intolerant and repressive. Hef was my friend. He was the coolest. And the hippest. And at the same time, the squarest. While he transformed from an Illinois prude to the ultimate urban sophisticate, he never lost the Midwest values on which he was raised. His word was bankable. His heart was a well of emotion, open for all to see. He was the least prejudiced man I ever knew. He had deep pockets and long arms. I admired him differently at different times. When I was a kid, he was the embodiment of everything I wanted but seemed hopelessly out of reach: beautiful women, a kingly mansion, a bunny-emblazoned DC-9. He smoked a pipe and wore pajamas, both day and night. So did my uncle Mario, but he was a doctor and unfortunately wasn’t dating Barbie Benton. In those days, Hef’s name was synonymous with sin. For me, like most young American boys, getting my hands on a Playboy was the ultimate forbidden fruit … the joys of living before the internet. But in those days, I thought the interviews with Norman Mailer, the short stories by John Cheever or the poems by Allen Ginsberg were mere fillers that kept The Girls of the Big Ten from slamming directly into Miss November. In my 20s and 30s, when fortune smiled on me and I actually came to know Hef, spending time at the mansion, I admired his decency and his hospitality, how he made this Jewish kid from Miami Beach with a couple of movies under his belt feel as if he’d made it in a town that can be both magical and difficult. I also realized that by his fostering the literary and performing arts, by putting the wealth of his organization and the power of his convictions squarely on the side of civil liberties for all of our citizens, he was trying to make this country a better and fairer place. Now that he’s gone, I realize more than ever that he didn’t have to do those things. Many titans of industry have no interest in contributing to the Greater Good. Certainly Hef enjoyed money and what it could buy. But his most treasured acquisitions were friends of whom there are too many to count and causes that were unabashedly progressive — of which he was justifiably proud. My personal relationship with Hef actually started when I read that Brian Grazer was to produce a biopic about him. I sent Brian my classic 1970s playboy pinball machine with a note telling him that he’d found his director. After Brian capitulated, I had a series of meetings with Hef, desperately trying to crack how to tell the story, which is the odyssey of his life. The development process went through many incarnations — Imagine, Universal (with Robert Downey Jr. attached), Jerry Weintraub at Warners, and then, thankfully, it became available again. A dream came true: I bought it for RatPac. There will never be another Hugh Hefner, but getting to make a movie about his life is, I guess, the next best thing. The America into which Hugh Hefner was born was in many ways intolerant and repressive. He, among only a handful of men in our history, made it less so. It saddened him to see the pendulum swinging the other way. But Hef was the consummate optimist. I’m sure he passed on believing that the pendulum will swing back, that the progress he was committed to and facilitated will ultimately become a permanent part of our national character. A version of this story appears in the Oct. 4 issue of The Hollywood Reporter magazine. To receive the magazine, click here to subscribe. Every time I see the director Brett Ratner I remind him who I am. “Danielle Berrin, from The Jewish Journal.” For starters, I figure the lecherous lothario could use some help keeping tabs on all the women in his life. But second, I want to remind him that if he acts like a jerk, I could wind up writing something like this: I’ve been cornered downstairs in the gold lamé disco basement at Brett Ratner’s house and he’s hitting on me. That was the opening line of a profile I wrote of Ratner in October 2008. I worked hard to persuade him to interview with me, but not that hard, since the very first time I met him, at an event at American Jewish University, he gave me his phone number. My M.O. after that was turn his own trick right back on him: Don’t take ‘no’ for an answer. That line is part of the Brett-Ratner-rise-in-Hollywood mythology – and has been referenced countless times (see: Entourage, profiles etc.). But it’s also part of the mythology of Brett Ratner the man: He doesn’t take ‘no.’ He has no boundaries. He does whatever the heck he wants. It’s part of his big-Hollywood-director cache. And most of the time, he gets away with it. Not anymore. My guess is that if it were up to Brett Ratner The Man, and not Brett Ratner The Director, to get women to like him, he could have played The Forty-Year-Old-Virgin instead of Steve Carrell. And not because he’s modest and cute and shy; because he’s disrespectful and smarmy and childish. Every time I see Brett Ratner, he hits on me again. Though only after he insults me: “You’re still at the Jewish Journal? Your piece on me was supposed to advance your career.” (Editor’s note: It did. I got salaried and health insurance.) The last time I saw him, at the Museum of Tolerance dinner honoring Tom Cruise, he said, “You’re cute, but can you cook?” Months later, I texted him to ask for an interview about “Tower Heist” (which he did not grant) and to maybe do a live Q-and-A with me in advance of the Oscar cast he was supposed to produce. I asked if we could have lunch and talk it over. He only wanted to know if I still had a boyfriend. After all this, I figure, ‘This is his shtick. This is what he does. This is who he is.’ But when I read what he says about other women, how he humiliates them with the bully pulpit he doesn’t deserve – for example, he referred to Olivia Munn, who at the age of 30 has accomplished more than most, as a “whore” and revealed to Howard Stern that he forced Lindsay Lohan, who has enough problems, to get checked for sexually transmitted diseases before he would sleep with her—I get angry. Then I feel inclined to say things like this: If Lindsay Lohan was infected with the Ebola virus, Ratner would be lucky to get five minutes in a room with her. But admittedly, I have Ratner baggage. When I look back on my first big Hollywood interview, I remember sitting on Ratner’s couch, with a handful of people around, watching Albert Lamorisse’s The Red Balloon, and repeatedly removing Ratner’s hands from between my legs. I was a young, inexperienced journalist then, and told myself that getting the story was more important than my dignity. So in a big way, I’m grateful to Brett Ratner – because the very same profile that got me a full-time reporting job was also the one that taught me that no story is worth compromising my integrity. I should have slapped him and stormed from the house. It would have been cinematic. It is precisely because movies represent some imaginative ideal that the movie industry should have standards. Lots of directors get away with way worse than Ratner – Roman Polanski comes to mind, for example. Famous people tend to get away with things, because they’re famous and they can. Today was the first day Brett Ratner didn’t get away with it. Earlier today, I wrote that I didn’t care whether Ratner produced the Oscars or not, but in the dark of evening, long after the sun has set, I applaud the Academy for taking a stand – for standing for something. It was a Jewish thing to do. Noted Hollywood fauxteur Brett Ratner appeared on Attack of the Show to pimp his latest brain-smoothing studio confection, Tower Heist. After a comfortable amount of brown-nosing, host Kevin Pereira pointedly asked the director if rumors were true that a particularly unflattering passage in former co-host Olivia Munn's memoir, Suck It, Wonder Woman!: The Misadventures Of A Hollywood Geek, had referred to Ratner. (The story, as Munn recounts it, involved a major movie director whipping out his tiny penis without warning and proceeding to jerk off, while simultaneously eating cocktail-sauce-slathered jumbo shrimps with the other hand.) Ratner immediately knew which story Pereira was referring to, and offered this version of events. (It begins in the video below at 3:45.) "I used to date Olivia Munn, I'll be honest with everyone here. But when she was 'Lisa.' She wasn't Asian back then. She was hanging out on my set of After the Sunset, I banged her a few times, but I forgot her. Because she changed her name. I didn't know it was the same person and so when she auditioned for me for a TV show, I forgot her, she got pissed off, and so she made up all these stories about me eating shrimp and masturbating in my trailer. And she talked about my shortcomings." At which point Pereira added: "To be fair, you did ask for shrimp cocktail in the green room today." So draw your own conclusions. Movie director and shrimp-eating fapper, Brett Ratner, called into Howard Stern (via EW) yesterday to spit out a half-assed apology to GLAAD for saying “rehearsals is for fags” during a Q&A for Tower Heiest last week and he also admitted that he lied when he said that he “banged” Olivia Munn back when she wasn’t Asian. With the scent of curdled goat leche and shrimp shit wafting off of his finger tips, Brett picked up the phone and said this to Howard about Olivia: “She’s actually talented. The problem is I made her look like she’s a whore.” Brett kept backpedaling (unfortunately he didn’t backpedal off of a cliff) and gave GLAAD an “I’m Soooo SOWWY” hug for using the word fag. “I apologize for any offense my remarks caused. It was a dumb way of expressing myself. Everyone who knows me knows that I don’t have a prejudiced bone in my body. But as a storyteller I should have been much more thoughtful about the power of language and my choice of words.” Brett is producing the next Oscars and the president of that shit says that his comments were “dumb and insensitive” but “the apology [Ratner] gave I truly believe comes from his heart. If I didn’t believe it, I would do something about it. This is about integrity and honoring the Academy Awards, but we all make mistakes and I believe he didn’t mean it.” That apology came from Brett’s checking account, since he has a stupid movie to sell, and not his heart. That is the correction. But really. Brett Ratner is an exact stereotype of a sleazy, flea-dicked movie director who keeps a murphy bed in his office and honestly thinks he’s fooling a bitch with those tragic hair plugs, so none of us should strain our emotions by being shocked when words of doucheness fall out of his mouth. Allow me to put it more eloquently: Fuck him, fuck his awful movies and fuck his sweaty piss bag of a face. Seriously, I’d rather lick a condensation drop on an overheated piss bag than go see Tower Heist. (Yes, mom, that is my way of telling you that I will not see Tower Heist with you when I visit.) And Brett should call into Howard Stern again tomorrow to apologize for calling himself a storyteller. | Summary: Jared Leto will be playing Hugh Hefner in a biopic directed by Brett Ratner, according to the Hollywood Reporter. And let's just say that's not exactly a sentence making the internet happy right now. Ratner, who is also planning to reboot the talk show Playboy After Dark, which Hef hosted in the 1960s, definitely thinks it's a good idea- "When (Leto) heard I got the rights to Hef's story, he told me, 'I want to play him. I want to understand him.' And I really believe Jared can do it. He's one of the great actors of today"-but many, many others do not agree: The Los Angeles Times rounds up a number of hilarious tweets on the matter, like this one: "As America's conscience, Jimmy Kimmel needs to speak out tonight against Jared Leto playing Hugh Hefner in a Brett Ratner film." Sample headlines on the subject. From Silver Screen Beat: "Jared Leto will star in Brett Ratner's Hugh Hefner biopic that literally nobody asked for." From Vice: "Jared Leto Is Going to Play Hugh Hefner in a New Biopic for Some Reason." A common thread running through the complaints is that everyone involved, particularly Hef and Ratner, is "sleazy." If you've forgotten why Ratner might be considered such, this interview in which he talked about "banging" Olivia Munn back when "she wasn't Asian" and this one in which he said he forced Lindsay Lohan to get checked for STDs before he would sleep with her might refresh your memory. He's been called "the epitome of casting couch" and "an exact stereotype of a sleazy... movie director who keeps a murphy bed in his office." (And then there was that gay slur he used.) As for what he thinks of Hef, Ratner penned a remembrance of him for Wednesday's issue of the Hollywood Reporter: "Hef was my friend. He was the coolest," it opens, and goes on to say that the Playboy founder "was trying to make this country a better and fairer place." Not everyone is upset; Men's Journal went with the headline "Ageless, Bearded Mountaineer Jared Leto to Star in Hugh Hefner's Biopic" and its article speculates Leto could be nominated for an Oscar for the role. | 3,844 | 528 | multi_news | en |
Summarize: This application is a division of U.S. patent application Ser. No. 104,789, filed Aug. 10, 1993, now U.S. Pat. No. 5,388,549. FIELD OF THE INVENTION This invention relates generally to milk sampling for diagnostic purposes, and more specifically to a method and apparatus for extracting a sample of milk to be tested from the milk line in a milking system, whilst substantially avoiding interruption of the routine milking procedure. BACKGROUND OF THE INVENTION Milking of an animal is effected by means of a claw having attached thereto cups which are connected to the animals teats. In the mechanised situation which exists in a milking installation, a milk line connects to the claw to receive the milk. In order to draw off the milk, a constant vacuum of about 0.5 Bar is applied to the milk line, switchable on and off by a valve. The vacuum is switched on prior to commencement of milking and connects the cups to the teats. After milking has been completed, the vacuum is switched off. A second vacuum system provides a pulsating vacuum to the outside of the liners, to stimulate milk flow and maintain blood circulation in the teats. It will be understood, therefore, that in the environment of a milking installation, there are generally available two sources of vacuum namely a constant or steady vacuum and a pulsating vacuum. THE INVENTION According to one aspect of the present invention, there is provided a method of extracting a sample of milk from a milk line to which a vacuum is applied, for extracting milk from an animal, according to which a portion of the milk is a milk/air mixture flowing in the milk line is diverted through a by-pass, in which is provided an extraction means operable by at least one of a fixed vacuum and a pulsating vacuum for separating from the milk flowing in the by-pass a sample thereof and delivering it to a testing device. The said extraction means may also serve to control activation of the testing device for the purpose of testing the extracted milk sample. Initiation of an operating cycle to the sample extracting means may be effected manually. Preferably, in an upstream part of the sample extracting means, the milk flowing in the by-pass is raised to a pressure slightly above atmosphere pressure. This is desirable in order to cause the sample of milk to be delivered to the testing device, notwithstanding the vacuum which is appplied to said by-pass to cause milk to be drawn through the milk line when said means is not delivering a sample. Preferably, the operating cycle of the sample extracting means is timed, and milk is delivered to the testing device only for a portion of said operating cycle. Thus, according to another aspect of the invention, there is provided a method of extracting a sample of milk from a milk line to which a vaccuum is applied for extracting milk from an animal, according to which a portion of the milk flowing in the milk line is diverted through a by-pass, in which is provided an extraction means operable by at least one of a fixed vacuum and a pulsating vacuum for separating a sample from the milk flowing in the by-pass and delivering the sample to a testing device, the extraction means being operable over an operating cycle which is timed. When the sample extracting means also controls activation of the testing device, the latter may be activated for a subsequent portion of the operating cycle. Preferably, at the junction where the by-pass connects to the milk line, the line is so formed as to produce a reservoir of milk. In this way a constant flow of milk is generated through the by-pass. In a preferred method, at a delivery device from which milk is delivered to the testing device, when the delivery device is restored to open the by-pass to a through flow of milk, a temporary inflow of air through the delivery device caused by the vacuum applied in the by-pass, cleans the delivery device of residual milk. Preferably, when the milk line is cleaned by flushing with cleaning fluid following the completion of a milking operation, the cleaning fluid also passes through the by-pass to clean the sample extracting means and delivery device. The invention also relates to sample extracting apparatus for carrying out the above described method. According to another aspect of the invention there is provided apparatus for extracting a sample of milk from a milk line to which a vacuum is connectable, the milk line in use being connected to an animal being milked, comprising a sample extracting means for passing a flow of milk received from the milk line, said means comprising a pump for pressurizing milk and for operating a timer, and a sampling valve receiving the pressurized milk and for delivering a sample of milk to a diagnostic testing device, said sampling valve being operable under control of the timer. Preferably the milk pump and the sample valve are vacuum operated. Where a pulsating vacuum is available, the milk pump may to advantage be operated by the pulsating vacuum, whilst the sampling valve is preferably operated from a source of steady vacuum. The testing device may also be an actuable device for carrying out the diagnostic test, and this device also may be operable by a steady vacuum under control of the timer. If desired a plurality of testing devices for carrying out different diagnostic tests may be similarly operated and controlled. One such diagnostic test may be a test for progesterone content of the milk. A preferred pump is single acting reciprocating pump. The linear stroke of the pump piston is preferably converted to a rotary movement by a suitable transmission device, for example a double-ratchet mechanism which indexes a gear wheel on both the forward and reverse strokes of the pump. In a preferred embodiment, the timer is driven by the transmission device through reduction gearing. In the timer, one or more timing cams are driven through a clutch, such as a wrap spring clutch, which includes a manually operable release, eg. a pawl. When the pawl is released, as by a push-button, an operating cycle is initiated during which the timing cam or cams are permitted to perform one complete revolution. A camming surface on one of the timing cams mechanically controls a valve which opens a steady vacuum to the sampling valve for a portion of the operating cycle, thereby diverting the pressurized flow of milk through the sampling device to the testing device. During a later portion of the operating cycle, a camming surface on another timing cam may in similar manner cause activation of the testing device. Except when the sampling valve is operated, milk flows through the pump and the sampling valve. Thus, when the milk line is flushed following a milking operation, cleaning fluid also flows through the sampling means. In a preferred sampling system, the sampling means is connected as a by-pass to a primary milk line. Preferably, at the junction where the by-pass joins such milk line, a collector is provided in the milk line to collect and retain a reservoir of the flowing milk. In this way, milk flows continously through the by-pass, even though the primary milk line is erratically passing a flow of milk, froth and air. DESCRIPTION OF EMBODIMENTS The method and apparatus in accordance with the invention are exemplified in the following description, making reference to the accompanying drawings, in which: FIG. 1 is a block diagram of a sampling system; FIGS. 2 and 3 show a sampling pump and timer unit respectively in two side views; FIG. 4 shows a detail of the pump and timer unit; FIGS. 5 and 6 show a sampler valve, respectively in non-operated and operated conditions; and FIGS. 7 and 8 show an actuable testing device, respectively in elevated and plan views. FIG. 9 illustrates the milk collector of FIG. 1. Referring to FIG. 1, the sampling system is connected to a milk line 10 to which a vacuum is applied during milking and through which, in use, is flowing milk being collected from an animal being milked. In fact, in use the line 10 is passing a mixture of milk, froth and air. The sampling system is in the form of a by-pass connecting to the milk line at a collector 12, which is simply a portion fitted into the milk line having a depressed base, so as to form a reservoir for milk. By virtue of the collector 12, milk flows through the sampling system in the form of a continous stream. In addition to the collector 12, the sampling system includes a pump and timer unit 14 and a sampling valve 16. An actuable testing device 18 may also be regarded a part of the system. In FIG. 1, the solid black line indicates milk flow, the dotted line indicates an applied fixed vacuum, and the dash-dotted line indicates an applied pulsating vacuum. A pulsating vacuum is applied to operate the pump and timer unit. The timer controls the application of a steady vacuum for operating the sampling valve and the testing device. The pump and timer unit 14 as depicted in FIG. 3 comprises a pump on the left-hand side and a timer on the right-hand side. The pump comprises an upper chamber 20 in the upper part of which reciprocates a diaphragm-supported piston 22 under the action of a pulsating vacuum applied at the inlet port 2 4 and of a return spring 26. The pump also has a lower chamber 28 in which reciprocates a diaphragm-supported secondary piston 30. The lower chamber has a milk inlet 32 and a milk outlet 34, each associated with a one-way valve 36, whereby milk entering the pump, under the influence of the vacuum applied to the milk line, is raised in pressure to slightly above atmospheric pressure. The pressurized milk then passes to the sampling valve 16. As shown in FIG. 4, the piston 22 in the upper chamber 20 drives a double ratchet mechanism in the form of gear wheel 38 and oppositely acting pawls 40, whereby the linear motion of the piston is converted into a stepped rotary movement of the gear wheel. The gear wheel 38 drives the timer. Thus, reverting to FIGS. 2 and 3, the gear wheel 38 couples, through reduction gearing 42, 44 with a disc 46 forming part of a wrap spring clutch 48 carried by a shaft on which are also mounted two timing cams 50, 52. The camming surface 54 on one of these cams is visible in FIG. 2. The wrap spring clutch also includes a pawl 56 normally engaging with a step in the periphery of the disc 46, therby to prevent rotation of said disc, whereby the timing cams are also held against rotation. However, the pawl 56 can be lifted by, for example, a manually depressed push-button 58, thereby causing the clutch spring to tighten and, start an operating cycle of the timer in which one complete revolution of the shaft carrying the disc and the timing cams takes place. In practice, such an operating cycle occupies about 300 strokes of the pump, taking about 5 minutes. The timing cams mechanically control two valves, one of which is shown in FIG. 2, one controlling application of the steady vacuum to the sampling valve 16 and one controlling application of the steady vacuum to the testing device 18. The sampling valve is shown in FIGS. 5 and 6, and comprises a valve member 60 having a head 62 in a milk chamber 61 which passes milk received from the pump, the milk entering at port 65 and leaving at port 67. The valve member 60 is operable by movement of a diaphragm acting thereon via member 63. Thus, when the timing cam 50 causes the steady vacuum to be applied at the inlet port 64, the valve member 60 is lifted for a short period during which a sample of milk is delivered from outlet 66, from the milk chamber, to the testing device. When the steady vacuum is withdrawn from the inlet port 64, spring 68 returns the valve member to the closed position. It is to be noted that, except when the valve member is operated, there is a through-flow of milk through the milk chamber back to the milk line so that the milk delivered to the testing device is representative of the milk flowing through the milk line at that instant. It also follows that, when the system is flushed with cleaning fluid after completion of a milking Operation, the sample valve is also washed clean. Moreover, when the valve member is restricted to close the milk sample outlet, a momentary back surge of air occurs through said outlet, due to the applied vacuum in the milk line, which sucks any milk clinging to the mating surfaces of the valve member and valve seat back into the milk chamber. The diagnostic testing device per se forms no part of the present invention. As illustrated in FIGS. 7 and 8, however, it comprises upper and lower relatively rotatable cups, respectively 70 and 72, with openings in the base of the upper cup which can be aligned with or closed off from reagent chambers in the lower cup. The milk sample is retained in the upper cup until the cups are relatively rotated to release some of the milk sample into the reagent chambers. Relevant to the present invention is the actuator means for effecting the necessary relative rotation of the cups. This occurs after the milk sample has been delivered to the upper cup from the sampling valve, which is ensured by appropriate positioning of the camming surface on the timing cam 52 relative to that of the camming surface in the timing cam 50 which controls the sampler valve. As shown in FIG. 8, in particular, the actuator means comprises a ratchet 74 on a cylindrical member 76 which is sealingly slideable relative to a fixed cylindrical member 78 under the influence of the steady vacuum, when applied at inlet port 80. Ratchet 74 drives a gear 82 carried by the rotatable cup. Restoration of the member 76 is by means of an internal spring 84. It will be appreciated that, by providing more timing cams on the timer of FIGS. 2 and 3, it is possible to actuate more than one actuable testing device, for carrying out different diagnostic tests, or to actuate a testing device requiring more than one actuation. Various modification of the above-described and illustrated arrangement are possible within the scope of the invention hereinbefore defined. In particular, for carrying out the method generally illustrated in FIG. 1, various other constructions of pump, timer and sampling valve, operable by steady and/or pulsating vacuum, may be employed. FIG. 9 illustrates the milk collector 12 of FIG. 1. Milk 13 collects in the depression 15 and is drawn off along tube 17 to the pump and timer unit 14. | Summary: Methods and apparatus enabling a diagnostic test to be carried out on milk flowing in a milk line from an animal being milked, without interrupting the milking process, in which a portion of the milk is diverted through a by-pass in which an extracting means (14) in the form of a vacuum-operable pump and timer acts to separate from the milk flowing in the by-pass a milk sample and to deliver said sample through an associated sampling valve (16) to a testing device (18). | 3,360 | 113 | big_patent | en |
Write a title and summarize: Previously, we proposed a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine as chronic nonspecific multiple ulcers of the small intestine (CNSU). By whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, we found four candidate mutations in the SLCO2A1 gene, encoding a prostaglandin transporter. The pathogenicity of the mutations was supported by segregation analysis and genotyping data in controls. By Sanger sequencing of the coding regions, 11 of 12 other CNSU patients and 2 of 603 patients with a diagnosis of Crohn’s disease were found to have homozygous or compound heterozygous SLCO2A1 mutations. In total, we identified recessive SLCO2A1 mutations located at seven sites. Using RT-PCR, we demonstrated that the identified splice-site mutations altered the RNA splicing, and introduced a premature stop codon. Tracer prostaglandin E2 uptake analysis showed that the mutant SLCO2A1 protein for each mutation exhibited impaired prostaglandin transport. Immunohistochemistry and immunofluorescence analyses revealed that SLCO2A1 protein was expressed on the cellular membrane of vascular endothelial cells in the small intestinal mucosa in control subjects, but was not detected in affected individuals. These findings indicate that loss-of-function mutations in the SLCO2A1 gene encoding a prostaglandin transporter cause the hereditary enteropathy CNSU. We suggest a more appropriate nomenclature of “chronic enteropathy associated with SLCO2A1 gene” (CEAS). The use of capsule endoscopy and balloon endoscopy has provided a better understanding of the features of small bowel ulcers among various gastrointestinal disorders, such as Crohn’s disease (CD), intestinal tuberculosis, and nonsteroidal anti-inflammatory drug (NSAID) –induced enteropathy [1,2]. Previously, we proposed a rare clinicopathologic entity characterized by multiple small intestinal ulcers of nonspecific histology and chronic persistent gastrointestinal bleeding as chronic nonspecific multiple ulcers of the small intestine (CNSU) [3,4]. The macroscopic findings of CNSU are characterized by multiple thin ulcers in a linear or circumferential configuration and concentric stenosis, and apparently mimic those of NSAID-induced enteropathy [4–7]. CNSU predominantly occurs in females and the symptoms, such as general fatigue, edema, and abdominal pain, appear during adolescence. The clinical course of the disease is chronic and intractable with reduced effects of immunosuppressive treatment including prednisolone and azathioprine. Although CNSU predominantly occurs in females, it also appears to be an autosomal recessive inherited disorder because of frequent parental consanguinity [8]. To identify the causative gene for this disorder, we performed whole-exome sequencing and identified recessive mutations in the SLCO2A1 gene, encoding a prostaglandin transporter, as causative variants. Furthermore, we replicated our findings in other patients with CNSU and established a genetic cause for this inherited disease. We performed whole-exome sequencing in five affected females with CNSU (A-V–2, B-IV–3, C-IV–3, D-II–4, and D-II–5) and one unaffected individual (A-V–3) (Figs 1 and 2). Parental consanguinity was noted in families A, B, and C. The average depth of sequence coverage in the whole-exome sequencing data was 68. 9× (S1 Table). We identified a total of 368,403 variants, of which 20,271 were non-synonymous or splice-site mutations. By filtering the data with dbSNP135, we found 2,406 variants located in 1,578 genes. Based on the parental consanguinity of the patients, we focused on the shared genes with homozygous variants among three affected individuals (A-V–2, B-IV–3, and C-IV–3) and found nine candidate genes, PCSK9, ASPM, DAG1, SLCO2A1, MCPH1, EFEMP2, DDHD1, PKD1L3, and SYNGR1. After consideration of the results for the unaffected individual (A-V–3) and another two sibling patients (D-II–4 and D-II–5), only SLCO2A1 remained as a candidate gene. The three patients with parental consanguinity (A-V–2, B-IV–3, and C-IV–3) had a homozygous splice-site mutation in the SLCO2A1 gene, c. 1461+1G>C or c. 940+1G>A (Fig 1 and Table 1). The two sibling patients had compound heterozygous mutations, c. 664G>A (p. Gly222Arg) and c. 1807C>T (p. Arg603X). All four mutations were predicted to be loss-of-function or damaging mutations by SIFT and PolyPhen–2. The four identified SLCO2A1 mutations were confirmed to be present in five affected individuals (A-V–2, B-IV–3, C-IV–3, D-II–4, and D-II–5) by Sanger sequencing (Fig 1). Segregation analysis of patient A-V–2 revealed that her unaffected parents, sister, brother, daughter, and son carried the heterozygous c. 1461+1G>C mutation (Fig 1A). To compensate for bias in our analysis, such as the possibility of ethnic-specific variants, we genotyped the four candidate variants in 747 unaffected Japanese subjects from our previous genome-wide association study [9]. All mutations except for the c. 940+1G>A mutation were absent in controls (Table 2). The c. 940+1G>A mutation was found in the heterozygous state in 3 of 747 controls, showing a similar allele frequency of 0. 0022 to the public exome database for the Japanese population (HGVD database). Subsequently, we screened all 14 coding exons and intron-exon boundaries using Sanger sequencing in 12 other CNSU patients, and identified two novel mutations, c. 421G>T (p. Glu141X) and c. 1372G>T (p. Val458Phe) (Table 1). Eleven of the 12 patients were found to have homozygous (nine patients) or compound heterozygous (two patients) SLCO2A1 mutations that were rare and predicted to be deleterious by SIFT, PolyPhen–2, and PROVEAN (Table 1). The remaining patient (66-year-old female), who did not have an SLCO2A1 mutation, was diagnosed as CNSU because of multiple ulcerations in the duodenum and jejunum. Although anti-tumor necrosis factor-α antibody therapy was ineffective, clinical improvement was achieved by enteral nutrition. Because CNSU can be misdiagnosed as CD in some cases, we searched for the six identified mutations of SLCO2A1 in CD patients to identify concealed CNSU patients. Among 603 patients previously diagnosed as CD [10], two individuals (patients 17 and 18 in Table 1) were found to carry a pair of compound heterozygous SLCO2A1 mutations, c. 940+1G>A/c. 547G>A and c. 940+1G>A/c. 421G>T, respectively. The c. 547G>A mutation (p. Gly183Arg), was a novel mutation at a highly conserved site and predicted to be deleterious by in silico analysis. The clinical information for the two individuals was reviewed retrospectively, and the diagnosis of CNSU was confirmed. In total, we identified seven deleterious SLCO2A1 mutations in 18 patients (Table 2). In total, we found 18 patients with CNSU confirmed by genetic analysis (Table 1); 14 of them were female. In all patients, the ulcers occurred in the ileum (Fig 2A–2D). The stomach and duodenum were affected in five (27. 8%) and eight (44. 4%) patients, respectively. Because mutations in the SLCO2A1 gene, encoding a prostaglandin transporter, have been reported to be the pathogenic cause of primary hypertrophic osteoarthropathy (PHO; OMIM 614441) [11–13], we investigated whether CNSU patients had clinical manifestations of PHO. Although no patients had any clinical manifestations of PHO requiring treatment, mild digital clubbing or periostosis was present in seven of 18 patients (S2 Table). Moreover, three male patients (patients 12,16, and 17) fulfilled the major clinical criteria for PHO, having digital clubbing, periostosis, and pachydermia. There were no female patients who fulfilled the major clinical criteria (Fig 2E and 2F). Among the identified SLCO2A1 mutations, a splice-site mutation of intron 7 (c. 940+1G>A) was the most frequent, and nine of the 18 patients were homozygous for this mutation. There were no obvious correlations between the types of mutations and the clinical phenotypes. Because the SLCO2A1 gene encodes a prostaglandin transporter that mediates the uptake and clearance of prostaglandins, the urinary levels of prostaglandin E metabolite (PGE-M) were measured. The urinary PGE-M levels in CNSU patients were significantly higher than those in unaffected individuals (p = 0. 00013; S1 Fig). Using RT-PCR, we demonstrated that splicing of the SLCO2A1 mRNA, derived from biopsy specimens of the small intestine, was altered in affected siblings with the homozygous c. 940+1G>A mutation (patients 6 and 7) compared with a control individual (Fig 3A). Sequencing of the RT-PCR products revealed deletion of the whole exon 7 of SLCO2A1, leading to a frameshift at amino acid position 288 and introduction of a premature stop codon after six amino acid residues (p. R288Gfs*7). Sequencing of the RT-PCR products of the transcripts in peripheral blood mononuclear cells from patient A-V–2 revealed that the homozygous c. 1461+1G>C mutation led to a 23-bp frameshift insertion into intron 10, resulting in a premature stop codon (p. I488Lfs*11) (S2 Fig). For functional analysis of the intact and truncated SLCO2A1 proteins, we investigated the 3H-labeled prostaglandin E2 (PGE2) transport ability in HEK293 cells transfected with intact SLCO2A1 and mutant SLCO2A1 proteins for each identified mutation (c. 940+1G>A, p. Gly222Arg, p. Arg603X, p. Glu141X, p. Val458Phe, and p. Gly183Arg). HEK293 cells transfected with intact SLCO2A1 showed the ability for PGE2 transport. In contrast, HEK293 cells transfected with the mutant SLCO2A1 proteins were unable to uptake 3H-labeled PGE (p < 0. 0001; Fig 3B). These findings demonstrated that the mutant SLCO2A1 proteins identified in patients lost their functional ability as a PGE transporter. In control sections of normal small intestinal mucosa, SLCO2A1 was expressed on the cellular membrane of vascular endothelial cells in the small intestine, as evaluated by immunohistochemistry and immunofluorescence staining with a specific anti-SLCO2A1 antibody recognizing the fifth extracellular domain coded by exons 9–11 of the SLCO2A1 gene (Fig 3C). We then analyzed the expression of SLCO2A1 in the small intestine of affected individuals with the homozygous c. 940+1G>A mutation (patients 6 and 7) by immunofluorescence staining. However, the immunofluorescence staining did not detect any SLCO2A1 protein in the vascular endothelial cells of the patients (Fig 3C). These results indicated that the entire SLCO2A1 protein was unexpressed in affected individuals with the homozygous c. 940+1G>A mutation, consistent with the results of the mRNA transcript sequencing. To investigate the subcellular localization of SLCO2A1 and the truncated SLCO2A1 protein (ΔSLCO2A1) corresponding to the c. 940+1G>A mutation, we constructed expression vectors for GFP-SLCO2A1 and GFP-ΔSLCO2A1 fusion proteins and transfected them into HEK293 cells. GFP-SLCO2A1 was localized on the cellular membrane (Fig 3D, arrows) as well as in the cytoplasm of transfected HEK293 cells, while GFP-ΔSLCO2A1 did not accumulate on the cellular membrane (Fig 3D). In this study, we performed whole-exome sequencing in five Japanese patients with CNSU and one unaffected individual, and identified the SLCO2A1 gene as the candidate for this disorder. We further confirmed that SLCO2A1 gene mutations were involved in the pathogenesis of CNSU by genotyping of control subjects and other CNSU patients. Moreover, a genetic analysis of 603 patients previously diagnosed as CD revealed that two CNSU patients had been included in this disease group. In total, we identified seven different mutations in the SLCO2A1 gene, comprising two splicing-site mutations, two truncating mutations, and three missense mutations, as the causative gene defects for CNSU. Therefore, we propose a more appropriate nomenclature, “chronic enteropathy associated with SLCO2A1 gene” (CEAS), for this disease. The SLCO2A1 gene encodes a prostaglandin transporter that may be involved in mediating the uptake and clearance of prostaglandins in numerous tissues [14–16]. This gene has already been reported as a causative gene for a subtype of PHO [11]. In fact, three of the seven identified mutations, c. 664G>A, c. 940+1G>A, and c. 1807C>T, have also been reported as causative mutations for PHO [11–13,17,18]. We found that three male patients with CEAS had all of the major clinical features of PHO, such as digital clubbing, periostosis, and pachydermia. Moreover, either digital clubbing or periostosis was present in seven of 18 patients. These findings indicate that CEAS and PHO share a causative gene and that their clinical features are profoundly influenced by other modifiers. Taken together with the facts that that CEAS predominantly occurs in females and PHO predominantly occurs in males [8,17], a sex-influenced gene or hormone may be the main disease modifier. Zhang et al. [17] reported that two female family members of a PHO patient had no clinical features of PHO, despite having a homozygous SLCO2A1 mutation. Moreover, it is interesting to note that these two siblings both had anemia and hypoalbuminemia, suggesting that they had CEAS. PHO is also known to be caused by mutations of HPGD, encoding 15-hydroxyprostaglandin dehydrogenase (15-PGDH), as well as SLCO2A1 [19]. The transmembrane prostaglandin transporter encoded by the SLCO2A1 gene delivers prostaglandins to cytoplasmic 15-PGDH, resulting in their degradation [14,20]. Because 15-PGDH is the main enzyme for prostaglandin degradation, systemic PGE2 levels are increased in patients with HPGD deficiency. Consistent with the findings in our present investigation, Zhang et al. [11] reported that the urinary levels of PGE2 and PGE-M in SLCO2A1-deficient individuals with PHO are significantly higher than those in controls. In fact, the clinical features of PHO were assumed to be caused by excessive PGE2. Meanwhile, although elevated levels of PGE2 in gastrointestinal tissues are commonly known to protect against mucosal inflammation via the prostaglandin receptor EP3/EP4 [21–23], multiple intestinal ulcers occur in CEAS. This discrepancy and the pathogenesis of intestinal ulcers need to be clarified in future studies. Although CEAS is presumed to be unaccompanied by immunological inflammation in its pathogenesis, a portion of CEAS patients can be misdiagnosed as CD because of the shared common clinical features, such as multiple small intestinal ulcers, anemia, and hypoalbuminemia. In this study, two of 603 patients previously diagnosed as CD were found to be affected with CEAS by genetic analysis. Because corticosteroid and anti-tumor necrosis factor-α antibody therapies are ineffective for CEAS, recognition and precise diagnosis of CEAS are critical to avoid unnecessary therapies. The findings of our investigation lead us to conclude that genetic analysis in addition to detailed clinical information including digital clubbing, blood tests, and gastrointestinal examinations are invaluable for distinguishing CEAS from CD. Cases of a similar enteropathy referred to as cryptogenic multifocal ulcerous stenosing enteritis (CMUSE) have been reported in Western populations [24–26]. This enteropathy has been shown to be an autosomal recessive inherited disease caused by mutations in the PLA2G4A gene [27]. CEAS and CMUSE share common clinicopathologic features with respect to age of onset, chronic and recurrent clinical course, and nonspecific stenosing small intestinal ulcers [4,25]. However, the sex predominance, response to steroid therapy, and lesion sites are obviously different between the two conditions. The PLA2G4A gene encodes cytoplasmic phospholipase A2-α (cPLA2α), which catalyzes the release of arachidonic acid from membrane phospholipids. CMUSE patients with compound heterozygous mutations of PLA2G4A have been reported to show globally decreased production of eicosanoids such as PGE2 and thromboxane A2, resulting in multiple ulcers of the small intestine and platelet dysfunction [27,28]. Because impaired prostaglandin use underlies CEAS, CMUSE, and NSAID-induced enteropathy, we propose a new entity of gastrointestinal disorders, namely “prostaglandin-associated enteropathy”. In conclusion, we have identified loss-of-function mutations in the SLCO2A1 gene as the cause of CEAS. The present findings clearly indicate that CEAS is a genetically distinct entity independent of other gastrointestinal disorders including CD, NSAID-induced enteropathy, and CMUSE. Further studies are needed to elucidate the pathogenesis of CEAS and identify new therapeutic molecular targets for “prostaglandin-associated enteropathy”. Written informed consent for genetic studies was obtained from each individual. The study was approved by the institutional review board at each collecting site in accordance with the Declaration of Helsinki Principles. We obtained blood samples and family pedigrees from 17 Japanese patients with CNSU and eight unaffected family members in 15 families. The diagnosis of CNSU was based on the published clinical criteria and clinical courses (S3 Table) [8,29]. Genomic DNA samples from 747 participants in our previous genome-wide association study for ulcerative colitis [9] and 603 patients with CD [10,30] were used after excluding subjects who recalled their consent. DNA was extracted from peripheral blood using standard methods. Whole-exome sequencing in five affected individuals (A-V–2, B-IV–3, C-IV–3, D-II–4, and D-II–5) and one unaffected individual (A-V–3) was performed to identify candidate genetic variants (Fig 1). Genomic DNA was enriched using a TruSeq Exome Enrichment Kit (Illumina, San Diego, CA, USA) according to the manufacturer’s instructions, and paired-end sequencing was carried out with an Illumina HiSeq 2000 instrument. Reads were aligned to the human genome reference sequence (hg19 NCBI build 37. 1) and decoy sequences using BWA software [31]. Duplicate reads were removed with the Picard program (http: //picard. sourceforge. net/). Recalibration and realignment of the data were accomplished with Genome Analysis Toolkit (GATK) [32,33]. Single nucleotide variants and small insertions and deletions (indels) were identified by GATK Unified Genotyper. The effect of each missense mutation was predicted using SIFT (http: //sift. jcvi. org/) [34], PolyPhen–2 (http: //genetics. bwh. harvard. edu/pph2/) [35], and PROVEAN (http: //provean. jcvi. org/) [36]. To compensate for bias in our analysis, such as the possibility of ethnic-specific variants, we genotyped the four candidate variants identified by exome sequencing in 747 unaffected Japanese subjects by Sanger sequencing and restriction fragment length polymorphism analysis (S4 Table). For further validation, Sanger sequencing of all exons of the SLCO2A1 gene in other CNSU patients was performed using standard protocols. Finally, we genotyped the six identified mutation sites in the SLCO2A1 gene in clinically diagnosed CD patients, because CNSU can be misdiagnosed as CD. Urine samples were collected from 15 CNSU patients and 13 unaffected individuals. The PGE-M levels were measured in duplicate using competitive enzyme-linked immunosorbent assays (Cayman Chemical, Ann Arbor, MI, USA). We analyzed the exon 7 and exon 10 boundary mutations using RT-PCR to examine the effects of the splice-site mutations on SLCO2A1 transcription. Total RNA was extracted from biopsy specimens of the small intestine and peripheral blood mononuclear cells using a NucleoSpin RNA Kit (Macherey-Nagel, Düren, Germany) or PAXgene Blood RNA Kit (Qiagen, Hilden, Germany). cDNA was synthesized using a PrimeScript First Strand cDNA Synthesis Kit (TaKaRa, Otsu, Japan). The PCR products obtained from the cDNAs were sequenced (S5 Table). A full-length cDNA (NM_005630) expression vector and C-terminally GFP-tagged cDNA expression vector were purchased from OriGene Technologies (Rockville, MD, USA). To construct vectors carrying a mutated cDNA, a KOD -Plus- Mutagenesis Kit (Toyobo, Osaka, Japan) was used according to the manufacturer’s instructions. The expression vectors were amplified by inverse PCR with specific primer sets (S6 Table). The PCR products were self-ligated, and transformed into Escherichia coli chemically competent DH5α cells. To correct a frameshift in the downstream of exon 7, a C-terminally GFP-tagged cDNA expression vector with deletion of exon 7 was amplified again. On the day before transfection, HEK293 cells were trypsinized, counted, and plated onto 12-well plates at a density of 4×105 cells/well. The cells were transfected by adding a premixed solution containing 0. 4 μg of expression vectors and 2 μl of ScreenFectA (Wako, Osaka, Japan). After 24 hours of incubation, the medium was exchanged twice with warmed Waymouth’s medium (Life Technologies, Carlsbad, CA, USA), and the cells were incubated for 30 minutes at 37°C in uptake medium containing [5,6, 8,11,12,14,15-3H (N) ]-PGE2 (PerkinElmer, Waltham, MA, USA) at 0. 6 nM. The cells were washed four times with cold Waymouth’s medium, and lysed with 200 μl of RIPA Buffer (Thermo Fisher Scientific, Hemel Hempstead, UK) containing a protease inhibitor (Roche, Basel, Switzerland). The total protein concentration was quantified using a BCA Protein Assay Kit (Thermo Fisher Scientific). Next, 150 μl of cell lysate was mixed with 5 ml of MicroScint–20 (PerkinElmer), and scintillation counting was performed in a Tri-Carb 3100TR liquid scintillation spectrometer (PerkinElmer). Formalin-fixed paraffin-embedded tissues were sectioned at 3-μm thickness. After antigen unmasking in 10 mM sodium citrate buffer (pH 6) for 15 minutes at 121°C, the sections were blocked with Protein Block Serum-Free (Dako, Glostrup, Denmark) for 30 minutes at room temperature. The sections were then incubated with a diluted anti-SLCO2A1 antibody (HPA013742; Sigma-Aldrich, St. Louis, MO, USA; antigen sequence: PSTSSSIHPQSPACRRDCSCPDSIFHPVCGDNGIEYLSPCHAGCSNINMSSATSKQLIYLNCSCVTGGSASAKTGSCPVPCAH) overnight at 4°C, followed by MAX-PO (MULTI) (Nichirei, Tokyo, Japan) for 30 minutes at room temperature. DAB solution (Nichirei) was applied for color development. After the immunocytochemistry, the sections were counterstained with Mayer’s hematoxylin solution (Nichirei). For immunofluorescence, the sections were incubated with a primary antibody mixture of the anti-SLCO2A1 antibody (HPA013742) and an anti-VE-cadherin antibody (LS-B3780; LifeSpan BioSciences, Seattle, WA, USA) overnight at 4°C, followed by a secondary antibody mixture of Alexa Fluor 568-conjugated goat anti-rabbit IgG (H&L) antibody and Alexa Fluor 488-conjugated goat anti-mouse IgG (H&L) antibody (Life Technologies) for 30 minutes at room temperature. The stained sections were analyzed using an ECLIPSE TE2000-U (Nikon, Tokyo, Japan). For observation of HEK293 cells expressing GFP-fusion proteins, cells were fixed with 4% paraformaldehyde phosphate buffer solution (Wako) for 20 minutes, and then permeabilized with 0. 1% Triton X–100 (Sigma-Aldrich) in D-PBS (-) solution (Wako) for 20 minutes. Nuclei were stained with 16. 2 μM Hoechst 33342 (Life Technologies) in D-PBS (-) solution for 5 minutes. GFP and nuclei were visualized using a 40× objective on an LSM710 Laser Scanning Confocal Microscope (Carl Zeiss, Oberkochen, Germany). The chi-square test and Fisher’s exact test, where appropriate, were used to analyze categorical data. Student’s t-test was used to compare quantitative data between two groups. Dunnett’s method was used for multiple comparisons with a control group. The analyses were performed using JMP Pro statistical package 11. 2. 0 (SAS Institute, Cary, NC, USA). Values of p < 0. 05 were regarded as statistically significant. | Title: A Hereditary Enteropathy Caused by Mutations in the SLCO2A1 Gene, Encoding a Prostaglandin Transporter Summary: Advanced diagnostic innovations such as capsule endoscopy and balloon endoscopy have provided better understanding of endoscopic findings of small bowel diseases. However, it remains difficult to diagnose small intestinal diseases such as Crohn's disease, intestinal tuberculosis, and nonsteroidal anti-inflammatory drug-induced enteropathy by the endoscopic findings alone. We previously reported a rare autosomal recessive inherited enteropathy characterized by persistent blood and protein loss from the small intestine. This enteropathy has an intractable clinical course with ineffectiveness of immunosuppressive treatment. In this study, we identified recessive mutations in the SLCO2A1 gene, encoding a prostaglandin transporter, as causative variants of this disorder by exome sequencing of four families, and showed that this disease is distinct from Crohn's disease. We also showed that the mutations found in the patients caused functional impairment of prostaglandin E2 uptake within cells. The present findings suggest that genetic analysis together with detailed clinical information is invaluable for diagnosis of the disease, and that there may be a concept of enteropathy referred to as "prostaglandin-associated enteropathy", irrespective of ethnic background. | 6,546 | 286 | lay_plos | en |
Summarize: MANILA (Reuters) - Philippine law enforcement agencies still have no proof yet on allegations made by two government officials that human rights groups may have become the “unwitting tools” of drug lords, police and drug enforcement agency officials said on Tuesday. FILE PHOTO: Philippine President Rodrigo Duterte announces the disbandment of police operations against illegal drugs at the Malacanang palace in Manila, Philippines early January 30, 2017. REUTERS/Ezra Acayan/File Photo Rights groups have denounced President Rodrigo Duterte’s ferocious war on drugs, in which thousands of people have been killed, either by police or by shadowy, unidentified masked gunmen on motorcycles. The mercurial leader, who says he must be tough to protect the people from the scourge of drugs, has criticised rights groups by saying they were “trivialising” his campaign and unjustly blaming the authorities for bloodshed. “We have no (proof) right now,” national police spokesman Chief Superintendent John Bulalacao told reporters when asked about proof of links between drug syndicates and human rights groups. “We are still validating reports that drug syndicates might be using human right groups to discredit the efforts of the government,” he said. Philippine Drug Enforcement Agency spokesman Derrick Carreon said the reports that drug lords were using rights groups to attack the government’s anti-drug efforts were based on recent findings that would have to be investigated. “We still have to dig further on this,” he told a regular news briefing at the presidential palace. Carreon said more than 123,000 drug suspects had been arrested since July 2016, which he said showed anti-drugs operations were not about killings. He said law enforcement agencies welcomed any criticism from rights groups and allowed them to observe anti-drug operations to prove that everything was done according to the rule of law. “We have made adjustments on the ground in order that we would not be criticised negatively in the conduct of our anti-drugs operations,” Carreon said. Duterte’s spokesman and foreign secretary did not present any evidence when they told reporters drug lords were using human rights groups to undermine the policy, statements against which the Human Rights Watch protested and said were “shameful” and risked provoking violence. The anti-narcotics campaign has raised international alarm and drawn criticism from some U.N. representatives, including High Commissioner for Human Rights Zeid Ra’ad al-Hussein, who suggested recently that Duterte needed to see a psychiatrist. Police say they have killed nearly 4,200 drug suspects who were violently resisting arrest since the launch of the crackdown 20 months ago, which Duterte has vowed to pursue until he steps down in June 2022. Expand President Rodrigo Duterte’s official spokesperson Harry Roque at the House of Congress in Quezon City, Metro Manila, Philippines May 15, 2017. © 2017 Reuters Philippine presidential spokesman Harry Roque alleged on Monday that “some human rights groups have become unwitting tools of drug lords to hinder the strides made by the administration.” That echoed recent comments by Foreign Secretary Alan Cayetano equating efforts of some unnamed human rights organizations to stop President Rodrigo Duterte’s murderous “war on drugs” with “being used by drug lords.” Cayetano said that human rights organizations demanding accountability for the carnage of the anti-drug campaign that has killed more than 12,000 people since Duterte took office in June 2016 were doing so “for politics, for business.” He criticized rights groups for “pushing an advocacy, an ideology” not based on “impartial and independent investigation.” Such public statements are just the latest salvo in the government campaign of denial and distraction to dodge growing international outrage against Duterte’s “war on drugs.” But these allegations are more than just gratuitous slurs aimed at undermining the integrity of already beleaguered Philippine human rights activists pushing back against the Duterte government’s systematic attack on rule of law and its instigation and incitement of possible crimes against humanity. Publicly linking human rights groups with “drug lords” constitutes a sinister veiled threat in a country in which government-compiled “watch lists” of suspected drug users and drug dealers have been linked to many of the “drug war’s” thousands of victims. This is a familiar government tactic. Earlier this month the government put at grave risk more than 600 people – among them a United Nations human rights expert and dozens of leftist activists – by labeling them as members of the Communist Party of the Philippines (CPP) and its armed wing, the New People’s Army (NPA). Duterte and his minions are misguided if they believe that gross intimidation tactics can derail moves toward “drug war” accountability by the International Criminal Court and the UN. Instead, the government’s moves to put Filipino human rights activists in potential harm’s way through dangerous, baseless accusations will likely only reinforce international resolve to challenge Duterte’s rights violations. Metro Manila (CNN Philippines, March 27) — Local and international human rights groups called out Presidential Spokesperson Harry Roque for his claims they are being used by drug lords to fight the Duterte administration. International group Human Rights Watch said publicly linking human rights groups to drug lords is the government's "intimidation tactics" against them. "These allegations are more than just gratuitous slurs aimed at undermining the integrity of already beleaguered Philippine human rights activists pushing back against the Duterte government's systematic attack on rule of law and its instigation and incitement of possible crimes against humanity," the group said in a Tuesday statement. The Presidential spokesperson issued a statement Monday that the attacks against Duterte's war on drugs have been non-stop. "We therefore do not discount the possibility that some human rights groups have become unwitting tools of drug lords to hinder the strides made by the Administration," he said. Roque on Tuesday responded to HRW's statement. "(HRW) should therefore not feel alluded to, exaggerate and politicize the issue to get some media mileage and public attention." Roque added he is standing by his earlier statement that some non-governmental organizations have become tools of drug lords. Local rights group Karapatan, however, said Malacañang continues to "invent the most ludicrous of stories" and create the "wildest accusations." "Malacanang places the blame on human rights organizations for the Duterte administration's failure to curb the illegal drug problem and accuses them of smearing the country's reputation," their statement read. The local group added the Palace is either creating a scenario to justify the killing of activists or evading accountability from domestic and international rights laws. "Either way, whatever they're having in Malacanang will not be enough to numb them and enable their hallucinations in covering up the Duterte regime's crimes against the people," the statement read. Local and international groups have criticized Duterte's war against illegal drugs. While government data show more than 4,000 deaths in anti-illegal drug operations, human rights groups believe the number to be as high as 13,000. Philippine drug-enforcement officials said on Tuesday that nearly 4,100 people have been killed in shootouts with police since the start of President Rodrigo Duterte’s bloody antinarcotics campaign, a growing number that has attracted international condemnation. The drugs war launched by the increasingly authoritarian president after he took office in June 2016 has alienated Mr. Duterte from Manila’s traditional allies including the U.S., creating an opening for China to increase its military and economic influence in Southeast... | Summary: Human rights groups shining a spotlight on mounting deaths in the Philippines' drug war now have the added chore of denying the government's claims they're involved. Following one of the bloodiest weeks in President Rodrigo Duterte's crackdown on drugs-13 people were killed in a single night, per the Wall Street Journal-presidential spokesman Harry Roque on Monday said "some human rights groups have become unwitting tools of drug lords to hinder the strides made by the Administration," per CNN. The comment echoed Foreign Secretary Alan Cayetano previously accusing rights groups of "pushing an advocacy... for politics, for business." Philippine officials later announced almost 124,000 people have been arrested for drug-related offenses and nearly 4,100 people have been killed in shootouts with police since June 2016. Human rights groups, which believe the government tally omits thousands of deaths, say accusations against them show a disregard for international outrage surrounding Duterte's regime, under investigation by the International Criminal Court for crimes against humanity. Human Rights Watch on Tuesday said Roque's comment was "shockingly dangerous and shameful" and "aimed at undermining the integrity of [those]... pushing back against the Duterte government's systematic attack on rule of law." Local rights group Karapatan added the president's office continues to "invent the most ludicrous of stories" and create the "wildest accusations." Duterte denies wrongdoing, however, and a national police spokesman tells Reuters that authorities are investigating Roque's claims. "We have no [proof] right now," the officer says. | 1,742 | 364 | multi_news | en |
Summarize: TECHNICAL FIELD The present invention relates to the measurement of a threshold at which a person begins to recognize flicker. The present invention particularly relates to a measurement device and a measurement method that are capable of easily and objectively measuring a flicker perception threshold without arbitrariness or inclination; and that are applicable to the evaluation of human mental fatigue (hereunder, this may simply be referred to as “fatigue”). BACKGROUND ART The modern world is called a stressful society, and there has been a strong demand for a method for objective fatigue measurement or evaluation for everyday use. In the past, quantitative fatigue evaluation (flicker test) was performed based on a phenomena in which the perception of “flicker” upon observation of a blinking light source changes depending on the fatigue degree; in particular, a phenomena in which the blinking frequency (Critical Flicker Frequency: CFF), which denotes the capability of perceiving a flicker, decreases along with an increase of fatigue degree. Heretofore, quantitative evaluation of fatigue degree using a flicker test has been widely employed, particularly in the industrial hygiene field. However, because this flicker test requires a dedicated device equipped with an LED or the like, it was not suitable for a universal or everyday use device. Under such circumstances, the inventors of the present invention developed a technology for enabling a fatigue degree test, which is based on the same flicker perception principle as in the flicker test, but which uses an image-displaying device such as a CRT or a liquid crystal display (see Patent Literature 1 below). The development of such a device enables, in principle, easy fatigue measurement on a daily basis using easily accessible devices, such as mobile phones or personal computers. CITATION LIST Patent Literature Japanese Unexamined Patent Publication No. 2008-220639 SUMMARY OF INVENTION Technical Problem In the aforementioned method used in a hitherto known flicker test, the threshold of CFF or the like was measured by gradually increasing the light-blinking cycle of a flicker; in other words, gradually decreasing the blinking frequency, and making the test subject subjectively signal the perception of “flicker” using a push-button indicator or the like. FIG. 1 shows a concept of the aforementioned hitherto known technique for measuring a flicker threshold. In the known method, the frequency was monotonically changed, and the frequency at the time where the test subject subjectively signaled the perception of “flicker” using a push-button indicator was measured. The measured thresholds obtained from multiple tests were averaged to determine the flicker threshold. However, since this flicker-displaying test uses a monotonous blinking light condition, after several tests, the test subject becomes able to empirically predict the result; i.e., the test subject can empirically predict the timing of flicker perception during the test. In addition, since the test result relied on the subjective perception by the test subject, there was no evidence to show that the user actually perceived the flicker. Therefore, the test subject can arbitrarily control the test result by empirically adjusting the timing of pushing the button. Further, since there were no standards for true/false answers regarding “flicker perception,” the perception timing (timing of pushing a button) by the test subjects was often arbitrary, thereby varying the test results. Moreover, because this test tends to force the test subject to watch a flicker of an evidently unrecognizable frequency for a long time, the test time was prolonged; additionally, the fatigue level was often underestimated. As such, in the known measurement method, the timing of pushing the button is likely to be influenced by the arbitrariness and inclination of the test subject; therefore, in this method, it was difficult to perform an objective measurement of a flicker perception threshold. The present invention was made to solve the aforementioned problems of hitherto known technologies, and provides a device and a method for the measurement of flicker perception threshold; the device and the method being capable of objectively measuring an accurate flicker perception threshold while eliminating arbitrariness or inclination of the test subject, thereby accurately evaluating mental fatigue of human. Solution to Problem The objective of the present invention is accomplished by the following means. Specifically, a method (1) for measuring a flicker perception threshold according to the present invention is a method for measuring a flicker perception threshold using a device comprising an operation unit, a display screen, and a recording unit, the method comprising: a first step of alternately displaying on the display screen an ON-period image and an OFF-period image each having a plurality of regions, the ON-period image and the OFF-period image having a contrast difference only in one corresponding region selected from the plurality of regions; a second step of, when it is judged during display of the ON and OFF-period images that an operation in the operation unit that is made by a test subject in response to perception of a flicker does not properly specify the one corresponding region, increasing the contrast difference, and when it is judged that the operation in the operation unit properly specifies the one corresponding region, recording the contrast difference at the time in the recording unit as a measured contrast difference; and a third step of judging whether the measured contrast difference recorded in the recording unit is converged; when it is judged that the measured contrast difference is not converged, decreasing the contrast difference, and when it is judged that the measured contrast difference is converged, determining a convergence value of the measured contrast difference as information corresponding to the flicker perception threshold. A method (2) for measuring a flicker perception threshold according to the present invention is a method for measuring a flicker perception threshold using a device method for measuring a flicker perception threshold using a device comprising an operation unit, a display screen, and a recording unit, the method comprising: a first step of blinking one region selected from a plurality of regions of the display screen at a first blinking frequency, and blinking other regions of the display screen at a second blinking frequency that cannot be perceived by human, a second step of, when it is judged during the blinking that an operation in the operation unit that is made by a test subject in response to perception of a flicker in the display screen does not properly specify the one region, decreasing the first blinking frequency, and when it is judged that the operation in the operation unit properly specifies the one region, recording the first blinking frequency at the time in the recording unit as a measured blinking frequency; and a third step of judging whether the measured blinking frequency recorded in the recording unit is converged; when it is judged that the measured blinking frequency is not converged, increasing the first blinking frequency, and when it is judged that the measured blinking frequency is converged, determining a convergence value of the measured blinking frequency as information corresponding to the flicker perception threshold. A method (3) for measuring a flicker perception threshold according to the present invention is a method for measuring a flicker perception threshold using a device comprising an operation unit, a plurality of groups of one or more light-emitting elements disposed in a plurality of regions, and a recording unit, the method comprising: A first step of blinking one or more light-emitting elements disposed in a region selected from the plurality of regions at a first blinking frequency, and blinking other light-emitting elements at a second blinking frequency that cannot be perceived by human, a second step of, when it is judged during the blinking that an operation in the operation unit that is made by a test subject in response to perception of a flicker of the light-emitting elements does not properly specify the one or more light-emitting elements in the region, decreasing the first blinking frequency, and when it is judged that the operation in the operation unit properly specifies the one or more light-emitting elements in the region, recording the first blinking frequency at the time in the recording unit as a measured blinking frequency; and a third step of judging whether the measured blinking frequency recorded in the recording unit is converged; when it is judged that the measured blinking frequency is not converged, increasing the first blinking frequency, and when it is judged that the measured blinking frequency is converged, determining a convergence value of the measured blinking frequency as information corresponding to the flicker perception threshold. The above methods (1) to (3) may be arranged such that the plurality of regions are separated from each other. The above methods (1) to (3) may be arranged such that the one region selected from the plurality of regions represents a number or a graphic, and the step of specifying the region is a step of specifying the number or the graphic. A device (1) for measuring a flicker perception threshold according to the present invention is a device comprising an arithmetic processing unit, a display screen, an operation unit, and a recording unit, wherein: the arithmetic processing unit alternately displays on the display screen an ON-period image and an OFF-period image each having a plurality of regions, the ON-period image and the OFF-period having a contrast difference in only one corresponding region selected from the plurality of regions; when it is judged during display of the ON and OFF-period images that an operation in the operation unit that is made by a test subject in response to perception of a flicker does not properly specify the one corresponding region, the arithmetic processing unit increases the contrast difference, and when it is judged that the operation in the operation unit properly specifies the one corresponding region, the arithmetic processing unit records the contrast difference at the time in the recording unit as a measured contrast difference; and the arithmetic processing unit judges whether the measured contrast difference recorded in the recording unit is converged; when it is judged that the measured contrast difference is not converged, the arithmetic processing unit decreases the contrast difference, and when it is judged that the measured contrast difference is converged, the arithmetic processing unit determines a convergence value of the measured contrast difference as information corresponding to the flicker perception threshold. A device (2) for measuring a flicker perception threshold according to the present invention is a device comprising an arithmetic processing unit, a display screen, an operation unit, and a recording unit, wherein: the arithmetic processing unit blinks the one region selected from a plurality of regions of the display screen at a first blinking frequency, and blinks other regions at a second blinking frequency that cannot be perceived by human, when it is judged during the blinking that an operation in the operation unit that is made by a test subject in response to perception of a flicker in the display screen does not properly specify the one region, the arithmetic processing unit decreases the first blinking frequency, and when it is judged that the operation in the operation unit properly specifies the one region, the arithmetic processing unit records the first blinking frequency at the time in the recording unit as a measured blinking frequency; and the arithmetic processing unit judges whether the measured blinking frequency recorded in the recording unit is converged; when it is judged that the measured blinking frequency is not converged, the arithmetic processing unit increases the first blinking frequency, and when it is judged that the measured contrast difference is converged, the arithmetic processing unit determines a convergence value of the measured blinking frequency as information corresponding to the flicker perception threshold. A device (3) for measuring a flicker perception threshold according to the present invention is a device comprising an arithmetic processing unit, an operation unit, a plurality of groups of one or more light-emitting elements disposed in a plurality of regions, and a recording unit, wherein: the arithmetic processing unit blinks one or more light-emitting elements disposed in the region selected from the plurality of regions at a first blinking frequency, and blinking other light-emitting elements at a second blinking frequency that cannot be perceived by human, when it is judged during the blinking that an operation in the operation unit that is made by a test subject in response to perception of a flicker of the light-emitting elements does not properly specify the one or more light-emitting elements in the region, the arithmetic processing unit decreases the first blinking frequency, and when it is judged that the operation in the operation unit properly specifies the one or more light-emitting elements in the region, the arithmetic processing unit records the first blinking frequency at the time in the recording unit as a measured blinking frequency; and the arithmetic processing unit judges whether the measured blinking frequency recorded in the recording unit is converged; when it is judged that the measured blinking frequency is not converged, the arithmetic processing unit increases the first blinking frequency, and when it is judged that the measured blinking frequency is converged, the arithmetic processing unit determines a convergence value of the measured blinking frequency as information corresponding to the flicker perception threshold. The above device (1) to (3) may be arranged such that the plurality of regions are separated from each other. The above device (1) to (3) may be arranged such that the one region selected from the plurality of regions represents a number or a graphic, and the step of specifying the region is a step of specifying the number or the graphic. Advantageous Effects of Invention The present invention makes it possible to determine whether the reaction of the test subject upon perception of a visual stimulation is appropriate, and control the contrast difference (or normalized contrast) or frequency based on the determination result, thereby eliminating arbitrariness or inclination of the test subject. With this characteristic, the present invention more objectively measures a flicker perception threshold. Accordingly, it becomes possible to more objectively evaluate mental fatigue of human. In particular, the hitherto known test cannot prevent excessive fatigue or an accident due to fatigue, because the test subject can intentionally provide a false non-fatigue result even though they are actually tired. In contrast, since the present invention can prevent such an intentional false result, it is useful for self-health management, as well as for labor management or safety management of production workers. Moreover, by adopting a perception threshold determining method in which the value converges to the perception threshold in the variation control for a contrast difference (or normalized contrast) or a frequency, it becomes possible to more accurately determine a flicker perception threshold in a short time, compared to the known method. More specifically, the present invention reduces the test time, and thereby further increases the convenience of the test. BRIEF DESCRIPTION OF DRAWINGS FIG. 1 A drawing showing a concept of measurement of a flicker perception threshold in the hitherto known technology. FIG. 2 A drawing showing a normalized contrast and a contrast difference. FIG. 3 A block diagram showing a flicker perception threshold measurement device according to an embodiment of the present invention. FIG. 4 A drawing showing a concept of measurement of a flicker perception threshold according to the present invention. FIG. 5 A flow chart showing an operation of a flicker perception threshold measurement device according to a First Embodiment of the present invention. FIG. 6 A drawing showing changes in contrast difference. FIG. 7 A drawing showing an example of a displayed image. FIG. 8 A drawing showing examples of displayed image. FIG. 9 A flow chart showing an operation of a flicker perception threshold measurement device according to a Second Embodiment of the present invention. FIG. 10 A drawing showing a convergence state of a normalized contrast in measurement of flicker perception threshold of a typical test subject. FIG. 11 A graph showing a measurement result of a flicker perception threshold of a test subject. DESCRIPTION OF EMBODIMENTS Embodiments of the present invention are described below in reference to the attached drawings. Hereinafter, “fatigue” means mental fatigue, unless otherwise specified. First Embodiment When a display apparatus displays an image at a fixed refresh rate, the luminance ratio (L OFF /L ON ) of the image between the OFF state and the ON state is defined as a normalized contrast (see FIG. 2 ). When an image is continuously displayed while gradually decreasing the normalized contrast, for example, as shown in n 1 to n 3 in FIG. 2, the test subject perceives a flicker at a point where the normalized contrast becomes equal to or below a certain threshold. By making the test subject react to notify the perception using push-button operation or the like when the test subject subjectively perceives a “flicker,” it is possible to define this threshold as information corresponding to a flicker perception threshold. Moreover, when an image is continuously displayed while gradually increasing the normalized contrast, for example, from zero, the test subject stops perceiving the flicker when the normalized contrast becomes equal to or greater than a certain threshold. As above, by making the test subject react to notify the inability to perceive the flicker using a push-button operation or the like when the test subject becomes unable to perceive a “flicker,” it is possible to define this threshold as information corresponding to a flicker perception threshold. Further, this threshold decreases in proportion to an increase in fatigue level of the test subject. In the present embodiment, when an image is presented to a test subject, a visual stimulation is generated by turning the image on and off while inducing a contrast difference (an example is shown in ΔL 1 in FIG. 2 ) in a part of the image. The information regarding the part (hereinafter referred to as a visual stimulation region) of the image is recorded, and judgment is carried out as to whether the reaction of the test subject regarding the perception of a flicker is appropriate based on the information regarding the visual stimulation region; then, according to the true/false judgment of the reaction of the test subject, the visual stimulation region and the contrast difference are changed. Further, to change the visual stimulation region and the contrast difference, a perception threshold determination method in which the value converges to the perception threshold is employed. This eliminates arbitrariness and inclination of the test subject during the test, thereby enabling a measurement of flicker threshold in a short time based on objective flicker perception. The normalized contrast of two images, i.e., an image in the OFF state and an image in the ON state, is in inverse proportion to the contrast difference of the two images. In particular, when the contrast (or a luminance value) in the ON state is fixed, the relationship between the normalized contrast and the contrast difference is uniquely determined. As the normalized contrast increases, the contrast difference decreases; and as the normalized contrast decreases, the contrast difference increases. Therefore, in the present specification, a control to decrease the normalized contrast is equal to a control to increase the contrast difference. In other words, a control to increase the normalized contrast is equal to a control to decrease the contrast difference. More specifically, the two terms are interchangeable according to the circumstances. However, in this specification, the term “normalized contrast” is used in the comparison with the CCF threshold of the hitherto known flicker test apparatus, and “contrast difference” is used to more clearly explain the present invention. FIG. 3 is a block diagram showing a flicker perception threshold measurement device according to the First Embodiment of the present invention. The measurement device of the present invention comprises an arithmetic processing unit (hereinafter referred to as a CPU) 1 for controlling the entire apparatus; a nonvolatile read-only memory (hereinafter referred to as a ROM) 2 for storing a program, etc.; a volatile rewritable memory (hereinafter referred to as a RAM) 3 for temporarily storing data; a nonvolatile rewritable recording unit 4 for continuously storing data; a clock unit 5 ; an interface unit (hereinafter referred to as an IF unit) 6 for interfacing with external apparatuses; an internal bus 7 for exchanging data (including control information) between the units; a display unit 8 ; and an operation unit 9. For example, a known computer or mobile device (mobile phone, PHS, PDA, etc.) can be used as the present measurement device of the present invention. The operation unit 9 includes operating means such as keys or pads. The display unit 8 includes a display screen (such as a liquid crystal display) and a drive unit for driving the display. The clock unit 5 is means for outputting current time information using an internal clock, such as a timer. The following briefly describes the operation of the present measurement device. The CPU 1 randomly specifies a predetermined region as region information in an image having two or more regions, stores the information in the recording unit 4, and generates two image data items according to previously specified conditions. These two image data items have a contrast difference in the respective aforementioned predetermined regions, and have the same contrast in the rest of the regions. The CPU 1 sends the two image data items to the display unit 8 via the IF unit 6, and displays the two images respectively in the ON and OFF times. The signal supplied to the display unit 8 is digital data or an analog video signal converted by the IF unit. The display unit 8 displays the received image data to a test subject 10 at a predetermined refresh rate. The test subject 10 observes the image presented in the display screen of the display unit 8, and operates the operation unit 9 to specify a certain region in the image when they recognize the appearance or disappearance of a flicker in the region. This operation information is sent to the CPU 1 via the IF unit 6, and is recorded in the RAM 3 or the recording unit 4. The CPU 1 carries out a judgment as to whether the region specified by the test subject is identical to the predetermined region stored as the region information. When they are identical, in other words, when the test subject properly specifies a flicker region, the CPU 1 generates an image again with a smaller contrast difference, and displays the image to the test subject. When they are not identical, in other words, when the test subject improperly specifies a flicker region, the CPU 1 generates an image again with a greater contrast difference, and displays the image to the test subject. As such, a judgment is carried out as to whether the test subject properly perceives a flicker by irregularly changing the image display state. In the present invention, instead of monotonously increasing or decreasing the normalized contrast between the images and storing the normalized contrast at the time when the test subject perceives a flicker, the normalized contrast (contrast difference) of the displayed images and a flicker region are irregularly changed, and the convergence value of the normalized contrast (contrast difference) at which the condition where the test subject perceives the appearance or disappearance of flicker is stably converged is determined as a flicker perception threshold. FIG. 4 is a drawing showing a concept of measurement of a flicker perception threshold according to the present invention. The operation of the measurement device according to the First Embodiment of the present invention is more specifically described below. FIG. 5 is a flow chart showing an operation of a measurement device according to the First Embodiment of the present invention. In the following description, all operations are explained as being carried out by the CPU 1, unless otherwise specified. It is also assumed that the CPU 1 reads out necessary data (including programs) as required from the ROM 2 and the recording unit 4, processes the data using a predetermined area of the RAM 3 as work area, and stores the temporary results and the final processing results in the recording unit 4, if necessary; the initial condition required for the measurement is stored beforehand in the recording unit 4. First, a menu is displayed on the display screen to allow the test subject 10 to select whether to carry out the measurement. When the test subject 10 operates the operating unit 9 to carry out the measurement, the following steps are performed. In Step S 1, the initial settings are made. For ease of explanation, the image contrast is explained as a luminance value. As the initial settings, the image-type information including the shape and size of the image having multiple regions, the ON period initial luminance value L ON, the initial luminance value difference (contrast difference) ΔL, and a time Δ for turning on and off an image in one type of contrast condition, are read out from the recording unit 4. The following example uses, as an example of the image, a circle consisting of four quarter-sector regions as shown in FIG. 6. In Step S 2, the various condition values for image data generation are calculated. More specifically, among the multiple regions, a flicker region to be displayed with a contrast difference is randomly determined, and this region is stored in RAM 3 as region information. For example, in the example of FIG. 6( a ), the right quarter-sector corresponds to the flicker region, and the upper, lower, and left quarter-sectors correspond to reference regions in which flicker is not generated. The reference regions have, for example, an intermediate luminance value. In this example, the positions of the upper, lower, left, and right quarter-sectors are stored in RAM 3 as region information, for example, and are associated with values from 1 to 4. Then, L 1 (=L ON ), L b (=L 1 −ΔL/2), L 2 (=L 1 −ΔL) are calculated, wherein L b represents the greater luminance value in the flicker region, L b represents the intermediate luminance value in the reference region, and L 2 represents the smaller luminance value in the flicker region. Finally, before starting the next Step S 3, time data is acquired from the clock unit 5. This time data is determined as a start time T. In Step S 3, an image is generated according to the conditions, such as the luminance value, determined in Step S 2, and the image is displayed on the display unit 8. In the example of FIG. 6( a ), a data item of an image having four regions with respective luminance values L b, L b, L b, and L 1 and a data item of an image having four regions with respective luminance values L b, L b, L b, and L 2 are alternately generated; and these images are alternately displayed in the display screen, corresponding to the ON and OFF states. In Step S 4, a judgment is made as to whether the test subject 10 operates the operation unit 9. For example, in Step S 3, a text message that reads “Press one of the keys ↑, ↓, ←, and → corresponding to the flicker region when flickering begins” or the like may be displayed on the display screen before the display of the image begins. If the test subject 10 presses a key as they perceive a flicker in one of the multiple regions of the displayed image, the sequence goes to Step S 7. If the key is not pressed by the test subject 10, the sequence goes to Step S 5. In Step S 5, the current time t is acquired from the clock unit 5 to be compared with the start time T. If the difference (t−T) is smaller than the time Δ (t−T<Δ), the sequence goes back to Step S 4. If the difference (t−T) is equal to or greater than the time Δ (t−T≧Δ), the sequence goes to Step S 6. As such, until one of the keys ↑, ↓, ←, and → is pressed by the test subject 10, the display is continued in the same contrast condition (luminance condition) for the time Δ. While judging that the test subject 10 is unable to perceive a flicker in the current contrast condition in Step 6, the sequence goes back to Step S 2 to start image display and perception in the new contrast condition. More specifically, if one of the keys is not pressed by the test subject 10 within a predetermined time, it is judged that the perception and operation are not properly performed. Accordingly, the contrast difference ΔL is increased by a predetermined value D 1. For example, in the example of FIG. 6( b ) on the condition ΔL=2×5=10, if the perception is not properly performed, the contrast difference is increased by 4(D 1 =4). As a result, the image generation, display, and perception are performed again in the condition ΔL=2×7=14 as shown in FIG. 6( c ). In Step S 7, a judgment is made as to whether the region indicated by the key pressed by the test subject 10 is identical to the region information stored in the RAM 3. When they are not identical, that is, when a wrong key is pressed, the sequence goes back to Step S 6. When they are identical, that is, when the right key is pressed, the sequence goes to Step S 8. In Step S 8, the contrast difference ΔL at the time is stored in the recording unit 4 as a measured contrast difference. Then, a judgment is made as to whether the difference between the measured contrast difference stored beforehand and the currently stored measured contrast difference is within a predetermined range. If, for example, the difference is within the predetermined range, the currently stored measured contrast difference is stored as a flicker perception threshold, and the sequence is ended. If the difference falls out of the predetermined range, the sequence goes to Step S 9. While judging that the fluctuation in flicker perception is too large in Step S 9, the contrast difference ΔL is decreased by a predetermined value D 2 (D 2 >D 1 ), and the sequence goes back to Step S 2 to start image display and perception in a new contrast condition. For example, when the perception is properly performed under the condition ΔL=2×10=20 in FIG. 6( a ), the contrast difference ΔL is decreased by 10(D 2 =10), i.e., the contrast difference ΔL is modified to the condition ΔL=2×5=10 in FIG. 6( b ), and the image generation, display and perception are started again with this condition. As explained above, through Steps S 1 to S 9, an image is presented to the test subject with a predetermined contrast condition during the time Δ, and a judgment is made to determine whether the operation by the test subject to notify the perception of a flicker is correct. By causing such irregular up-down variation in the contrast condition, more specifically, by varying the displayed image based on this true/false judgment, it is possible to gradually approximate the flicker perception of the test subject to a certain objective perception threshold, and thereby converge the threshold. In this manner, it is possible to objectively measure the flicker perception threshold of a specific test subject. Accordingly, it is possible to determine whether the test subject is experiencing fatigue by measuring and recording a flicker perception threshold when the test subject is free from fatigue; then measuring a flicker perception threshold again for the same test subject in the same manner, and comparing the obtained flicker perception threshold with the value obtained when the test subject was free from fatigue. The present invention is not limited to the above First Embodiment; and may be altered, for example, by varying the flow chart shown in FIG. 5. Specifically, although the above embodiment increases and decreases the contrast difference ΔL in two stages (D 2 >D 1 ) depending on the true/false judgment, for example, it is also possible to vary the contrast difference ΔL in more multiple stages, thereby controlling the contrast difference ΔL so that a large value is set in the beginning of the measurement and the value is gradually decreased as the measurement proceeds. As such, in addition to the up-down variation in the above embodiment, the contrast difference ΔL may also be controlled using other psychophysical methods for determining a perception threshold. In this case, the flicker threshold may be determined by averaging the values of measured contrast difference recorded in the several latest measurements. It is also possible to unify the variation amount of the contrast difference ΔL (i.e., D 1 =D 2, for example, 2), though it prolongs the measurement. The shape and size (number of pixels) of the image to be displayed may be arbitrarily determined. For example, as in the image shown in FIG. 7, the region corresponding to the number may be determined as the flicker region while determining the remaining area as a reference region. In this case, if the contrast difference is too small, the number cannot be identified. Therefore, in this case, by creating a visual stimulation using the contrast difference between the number and the background, and carrying out a judgment as to whether the test subject notifies the perception by pressing a key corresponding to the number, it is also possible to carry out the measurement of flicker perception threshold of a test subject as in the above embodiment. In this case, the value corresponding to the number shown in the display may be used as the region information. FIG. 8 shows another example. In this example, one of the seven regions of the image is determined as the flicker region, while determining the remaining area as a reference region. As in the First Embodiment above, by carrying out a judgment as to whether the test subject properly specifies the flicker region, it is possible to measure a flicker perception threshold of the test subject. Moreover, instead of the real-time image generation by the CPU 1, it is also possible to read out image data previously generated and stored in the recording unit 4, as required. Furthermore, although the contrast in the reference region is identical to the intermediate value between the two different contrasts in the above embodiment, it is also possible to perform the measurement of a flicker perception threshold as above using an arbitrary value for the contrast in the reference region. However, the contrast in the reference region is preferably identical to the intermediate value of the contrast difference. It is also possible to generate the contrast difference by changing the color of the displayed image. Further, in a method adopting a procedure to set a large initial contrast difference and then decrease the contrast difference, it is possible to determine the measured contrast difference at which the test subject stops perceiving the flicker as the flicker perception threshold. Second Embodiment The fundamental structure of the measurement device according to the Second Embodiment is the same as the measurement device ( FIG. 3 ) in the First Embodiment, except that the flicker display is performed in multiple regions of the display screen of the display unit 8 at different blinking frequencies. More specifically, instead of changing the contrast difference of an image to be presented to the test subject using a display screen, this device changes the blinking frequency. In the Second Embodiment, a visual stimulation is generated by blinking one of the regions in the display screen at a first blinking frequency that falls within a blinking frequency range perceivable by human. Further, the device also causes the remaining area of the display screen to blink at a second blinking frequency that falls within a blinking frequency range unnoticeable by human (including 0 Hz, at which the image is not blinked); more specifically, the device causes the remaining area of the display screen to blink at a blinking frequency, which appears as a continuous ON state. The device records the region information regarding the region (visual stimulation region) that blinks at the first blinking frequency, carries out a judgment as to whether the test subject properly reacts to the flicker perception based on the region information, and then varies the first blinking frequency and the blinking visual stimulation region in the image according to the true/false judgment regarding the reaction of the test subject. Further, to vary the visual stimulation region, a perception threshold determination method in which the value converges to the perception threshold is employed. This eliminates arbitrariness and inclination of the test subject during the test, thereby carrying out a measurement of flicker threshold in a short time based on objective flicker perception. The Second Embodiment is essentially based on the same control principle as that of the First Embodiment. Therefore, in the following explanation, only the steps different from those in the First Embodiment are described. The following briefly describes the operation of the measurement device according to the Second Embodiment. The CPU 1 randomly specifies a predetermined region as region information in an image having two or more regions and stores the information in the RAM 3, and then causes the predetermined region to blink at the first blinking frequency while causing the remaining area of the display screen to blink at the second blinking frequency. The test subject 10 observes the image displayed on the display screen of the display unit 8, and operates the operation unit 9 to specify a certain region in the image when they recognize the appearance or disappearance of a flicker in the region. This operation information is sent to the CPU 1 via the IF unit 6, and is recorded in the RAM 3 or the recording unit 4. The CPU 1 carries out a judgment as to whether the region specified by the test subject is identical to the predetermined region stored as the region information. When they are identical, in other words, when the test subject properly specifies a flicker region, the CPU 1 generates an image again by increasing the first blinking frequency, and displays the image to the test subject. When they are not identical, in other words, when the test subject improperly specifies a flicker region, the CPU 1 generates an image again by decreasing the first blinking frequency, and displays the image to the test subject. As such, a judgment is carried out as to whether the test subject properly perceives a flicker by irregularly changing the image display state. In the present invention, instead of monotonously increasing or decreasing the blinking frequency and storing the blinking frequency at the time when the test subject perceives a flicker as in the prior art, the first blinking frequency and a flicker region are irregularly changed, and the convergence value of the first blinking frequency at which the condition where the test subject perceives the appearance or disappearance of flicker is stably converged is determined as a flicker perception threshold. The operation of the measurement device according to the Second Embodiment of the present invention is more specifically described below. FIG. 9 is a flow chart showing an operation of a measurement device according to the Second Embodiment of the present invention. First, a menu is displayed on the display screen of the display unit 8 to allow the test subject 10 to select whether to carry out the measurement. When the test subject 10 operates the operating unit 9 to carry out the measurement, the following steps are performed. The following example uses the image shown in FIG. 7 that consists of a region representing a number and a background region, as an example of the flicker region. In Step S 21, the initial settings are made. Specifically, a variety of information including the type, shape and position of the flicker region, the initial value of the first blinking frequency f 1, the initial value of the variation range Δf of the first blinking frequency, the second blinking frequency f 2, and a time Δ as a duration for blinking a display screen with a certain blinking frequency condition are read out from the recording unit 4. Here, the initial value of f 1 may be f 2. In Step S 22, the various conditions for flicker display are calculated. More specifically, a single-digit number to be displayed at the first blinking frequency is randomly determined, and is stored in RAM 3 as region information. Then, first image data corresponding to the flicker region for showing the number, and second image data corresponding to the background region where no flicker is displayed are generated. Here, f 1 represents the first blinking frequency for the flicker region, and f 2 represents the second blinking frequency for the background region. Finally, before starting the next Step S 23, time data is acquired from the clock unit 5. This time data is referred to as a start time T. In Step S 23, according to the blinking conditions determined in Step S 22, the device causes the region corresponding to the randomly set single digit number to blink at the first blinking frequency f 1, and causes the background region to blink at the second blinking frequency f 2. More specifically, the first image data is displayed on the display screen at the first blinking frequency f 1, and the second image data is displayed on the display screen at the second blinking frequency f 2. In Step S 24, a judgment is made as to whether the test subject 10 operates the operation unit 9. For example, in Step S 23, a text message that reads “Press the key of the number shown” or the like is displayed on the display screen before the display screen starts blinking. If the test subject 10 presses one of the keys 0 to 9 as they perceive the flicker display of the number, the sequence goes to Step S 27. If the key is not pressed by the test subject 10, the sequence goes to Step S 25. In Step S 25, the current time t is acquired from the clock unit 5 to be compared with the start time T. If the difference (t−T) is smaller than the time Δ (t−T<Δ), the sequence goes back to Step S 24. If the difference (t−T) is equal to or greater than the time Δ (t−T≧Δ), the sequence goes to Step S 26. As such, until one of the keys is pressed by the test subject 10, the display is continued in the same blinking condition for the time Δ. While judging that the test subject 10 is unable to perceive a flicker with the current first blinking frequency f 1 in Step 26, the first blinking frequency f 1 is decreased by Δf and the sequence goes back to Step S 22 to start image display and perception in the new contrast condition. More specifically, if one of the keys is not pressed by the test subject 10 within a predetermined time, it is judged that the perception and operation are not properly performed. In Step S 27, a judgment is made as to whether the number indicated by the key pressed by the test subject 10 is identical to the stored region information. When they are not identical, that is, when a wrong key is pressed, the sequence goes back to Step S 26. When they are identical, that is, when the right key is pressed, the sequence goes to Step S 28. In Step S 28, the first blinking frequency f 1 at the time is stored in the recording unit 4 as a measurement frequency. Then, a judgment is made as to whether the difference between the measurement frequency stored beforehand and the currently stored measurement frequency is within a predetermined range. If the difference is within the predetermined range, for example, the currently stored measurement frequency is stored as a flicker perception threshold, and the sequence is ended. If the difference falls out of the predetermined range, the sequence goes to Step S 29. While judging that the fluctuation in flicker perception is too large in Step S 29, the first blinking frequency f 1 is decreased by Δf, and the sequence goes back to Step S 22 to start the blinking of the display screen of the display unit 8 in the new blinking condition to allow the test subject to perform the perception. As explained above, through Steps S 21 to S 29, an image is presented to the test subject in a predetermined flicker condition during the time Δ, and a judgment is made as to whether the operation by the test subject to notify the perception of a flicker is correct. With such irregular up-down variation of the first blinking frequency and irregular variation of the region that blinks at the blinking frequency based on this true/false judgment, it is possible to converge the flicker perception threshold to a certain objective perception threshold. In this manner, it is possible to objectively measure the flicker perception threshold of a specific test subject. The present invention is, of course, not limited to the above Second Embodiment; and may be altered, for example, by changing the flow chart shown in FIG. 9. Specifically, it is possible to vary the variation range Δf of the first blinking frequency; more specifically, it is possible to control Δf so that a large value is set in the beginning of the measurement and that the value is gradually decreased as the measurement proceeds. The value of f 1 may be converged using various psychophysical methods for determining a perception threshold. Further, the blinking region displayed in the display screen may be varied in many ways as shown in FIG. 6 and FIG. 8. Furthermore, when a control method in which a small first blinking frequency is set in the beginning of the measurement and the value is gradually increased, it is possible to determine the first blinking frequency at which the test subject stops perceiving the flicker as the flicker perception threshold. In addition, although the above Second Embodiment performs flicker display using a display screen, it is also possible to use a display device comprising a plurality of LED groups, thereby generating a flicker using these LEDs instead of a display screen. Each LED group may comprise single or plural LEDs. Further, although the CPU 1 generates the image data in real time in the above Second Embodiment, it is also possible to read out image data previously generated and stored in the recording unit 4 ; i.e., it is possible to read out a set of plural image data items showing a single-digit number, and a corresponding set of image data showing the background from the recording unit 4. Although the program for measuring the degree of mental fatigue is stored beforehand in the ROM 2 in the above First and Second Embodiments, it is also possible to download the program to the measurement device by accessing an Internet server, or installing the program in the measurement device via a detachable recording medium, such as a memory card. The features of the present invention are more specifically described below in reference to Examples. EXAMPLE 1 A personal computer (PC, hereinafter) having a liquid crystal screen that refreshes the display at 30 Hz was implemented with a program for carrying out the method of the present invention. Using the PC, image data was generated by temporally or spatially varying the normalized contrast of a stimulation object (image), thereby displaying a black background in the screen. An experiment was performed by carrying out a true/false judgment as to whether the test subject properly perceives the flicker, and finding the time point at which the perception by the test subject was stably ended. An image in which the four quarter-sectors are disposed in a circle having a 10-pixel diameter, as shown in FIG. 6, was used as the stimulation, i.e., the image to be presented to the test subject. When the test subject perceived a flicker, they pressed one of the arrow keys (↑, ↓, ←, →) corresponding to the flicker region. The time Δ, which is a predetermined time during which the presence or absence of push-button operation is judged, was 3 s (seconds). As shown in FIG. 2, the normalized contrast was L OFF /L ON, which is a ratio of the luminance value (L OFF ) of the OFF pixel to the luminance value (L ON ) of the ON pixel. Further, during the experiment, the luminance value L ON of the ON pixel was fixed to 255 (the maximum value for 8-bit). More specifically, the greater one of the luminance value (luminance value L 1 ) was fixed to 255. Further, the initial contrast difference ΔL was set to a luminance of 1 gradation. Accordingly, the initial intermediate luminance value L b in the three regions with no flicker was (L 1 −ΔL/2=255−½=) 254.5. The smaller one of the luminance values (luminance value L 2 ) was (L 1 −ΔL=255−1=) 254. Accordingly, the initial normalized contrast was about 0.996 (254/255=). With these conditions, a typical test subject performed a single operation of flicker perception threshold measurement. More specifically, with these initial conditions, an image was generated by temporally switching the luminance value of an arbitrarily-selected region between L 1 and L 1 −ΔL, and setting the luminance value of the other three regions to L 1 −ΔL/2; the generated image was presented to the test subject. When the test subject was unable to perceive a flicker within a predetermined time (3 s), or when the reaction (keystroke) of the test subject to notify the perception was judged to be improper, the normalized contrast was decreased by 1/255 (the contrast difference ΔL was increased by 1 gradation) so as to allow the test subject to more easily perceive a flicker, and an image of a new randomly-selected flicker region was generated and presented to the test subject. On the other hand, when the reaction (keystroke) of the test subject to notify the perception was judged to be proper within a predetermined time, the contrast difference ΔL was stored in the recording unit 4, the normalized contrast was increased by 1/255 (the contrast difference ΔL was decreased by 1 gradation) so as to make the flicker perception by the test subject more difficult; and an image of a new, randomly selected flicker region was generated and presented to the test subject. As such, the normalized contrast (i.e., contrast difference ΔL) and the flicker region were repeatedly changed according to the true/false judgment of the flicker perception by the test subject until the normalized contrast stably reached a certain threshold. FIG. 10 is a drawing showing a convergence state of the normalized contrast in a single measurement of flicker perception threshold of a typical test subject. The horizontal axis represents a measurement time (seconds) elapsed since the examination was started. The vertical axis represents the normalized contrast. As shown in FIG. 10, the normalized contrast moved from the upper left of the figure, where the flicker perception cannot be perceived, to the lower right, and gradually approached the flicker perception threshold of the test subject. The keystroke accuracy rate was determined to be 50/50 when the normalized contrast was about 0.957 and the contrast difference ΔL was 10.965. At this point, the measurement was ended, and it was judged that the perception was stably converged. Accordingly, in this measurement example, the flicker perception threshold of the test subject was determined to be 0.957. The time taken to the measurement was about 80 sec. Such measurement of the normalized contrast at which the test subject starts perceiving a flicker without arbitrariness or inclination enabled objective evaluation of the fatigue degree. Further, although it is not shown, the flicker perception threshold obtained for the same test subject was stable. This also confirmed that the present invention can eliminate arbitrariness in key operation of the test subject, unlike the hitherto known measurement device that cannot ensure stability of the obtained threshold. Moreover, in the hitherto known measurement device, the measurement, which took 20 to 30 s, was performed five times, and a flicker perception threshold was found by averaging the five measurement values; therefore, the entire measurement took 2 to 3 minutes for each test subject. In contrast, the measurement of the present invention takes about 60 to 90 sec., as is evident from the present example, in which it took 80 sec. As such, the measurement time was desirably reduced. EXAMPLE 2 To evaluate the reliability of the present invention, the measurement result of the present invention was compared with the measurement result of a hitherto known flicker measurement device. As in Example 1 above, the normalized contrast of the stimulation was temporally and spatially varied, and observation of how the normalized contrast (corresponding to the flicker value), which is a flicker perception threshold, changes depending on the fatigue load was performed. In this evaluation, the test subject had a fatigue load caused by all-night labor that continued from late evening to the next afternoon, and the normalized contrast of a stimulation (image) displayed on a display screen was varied using a PC, thereby measuring the flicker perception threshold. The evaluation is specifically described below. The test subject was a healthy adult. The test subject had designated intermittent daytime labor before the evaluation; therefore, the test subject already had a certain level of fatigue at the beginning of the evaluation. The first measurement was carried out at 2:30 a.m. The flicker perception threshold was measured using the respective measurement methods of the known flicker measurement device and the present invention. Thereafter, the test subject had 6 more hours of designated labor. The second measurement of flicker perception threshold was carried out at 8:30 a.m. using the two measurement methods. Thereafter, the test subject had a meal and a short sleep. After six hours, i.e., at 2:30 p.m., the third measurement of flicker perception threshold was carried out. The flicker perception threshold was measured using a standard flicker device and a PC in this order. As described above, the measurement was performed three times. The stimulation was presented in the same conditions as in Example 1 above. A MacBook Pro (Apple) was used as the PC. A Roken Digital Flicker Model RDF-1 (manufactured by Shibata Co., Ltd.) was used as the standard flicker device. A red stimulation target was displayed while decreasing the frequency from 55 Hz at a rate of 1 Hz per second. FIG. 11 is a graph showing a measurement result of a flicker perception threshold of a test subject. In this graph, the vertical axis presents the flicker perception thresholds normalized by the measurement values obtained at 2:30 p.m. More specifically, the measurement values obtained at 2:30 p.m. are set to 100, and the values obtained thereafter in proportion to 100 are plotted. The graph reveals that the flicker perception thresholds measured in the above two methods became lowest in the measurement at 8:30 a.m. when the test subject had maximum fatigue. The graph also reveals that, after the test subject had a short sleep, both of the flicker thresholds measured in the above two methods recovered and became higher than those measured at 2:30 a.m. Further, when the values measured in the two methods are normalized with their variation ranges, respectively, in other words, when the maximum value and the minimum value are plotted within the same width, the flicker thresholds show substantially the same tendency. Accordingly, it can be assumed that the changes in measurement values obtained by the standard flicker device were substantially duplicated in the measurement values obtained by a PC. As described, it was confirmed that the flicker perception thresholds measured in the above two methods were decreased by a fatigue load caused by all-night labor, and that the fatigue was recovered by taking a short sleep break. It was also shown that the flicker perception thresholds measured according to the method of the present invention reflected the fatigue conditions, as with the flicker values measured by the know method. INDUSTRIAL APPLICABILITY The present invention is able to objectively evaluate a flicker perception threshold and the fatigue of a test subject by using a computer or a mobile phone with ordinary functions, or by using a dedicated device. The present invention prevents arbitrary control of the measurement result, thereby obtaining an objective measurement result. With this advantage, the present invention is useful for appropriate health-care management and prevention of industrial accidents and injuries/illness at production sites. REFERENCE NUMERALS 1 Arithmetic processing unit (CPU) 2 Read-only memory (ROM) 3 Rewritable memory (RAM) 4 Recording unit 5 Clock unit 6 Interface unit (IF unit) 7 Internal bus 8 Display unit 9 Operation unit | Summary: The measurement method involves a step for displaying on a display screen an image in which one corresponding region exclusively that has been selected from regions provided has a contrast difference when on and when off; a step for enlarging the contrast difference in the image display in cases which an operation for an operation unit accompanying the flickering perception of a subject is assessed to not be correctly specifying the one aforementioned selected region, and, in cases which a correct specification has been assessed, recording the contrast difference at that point in a recording unit as the measurement contrast difference; and a step for assessing whether the aforementioned recorded measurement contrast difference has converged, reducing the contrast difference when convergence has not been assessed, and determining the convergence value of the aforementioned measurement contrast difference as information corresponding to the flickering perception threshold when convergence has been assessed. | 12,456 | 188 | big_patent | en |
Summarize: Back pay: John Zelepos, owner of Mystic Pizza, is being forced to pay back wages to 110 employees. A Connecticut restaurant owner will pay a total of more than $105,000 in back pay to 110 employees after cheating them out of wages for more than two years. The State of Connecticut Department of Labor began investigating Mystic Pizza owner John Zelepos after receiving three individual complaints from mis- or unpaid employees, NBC Connecticut reports. The investigation found that Zelepos reportedly has more than 150 minimum wage and overtime violations where employees received less than minimum wage or did not receive required overtime pay. The DOL reports that Zelepos paid employees working as hosts, cooks, and dishwashers as little as $5.69 an hour. Others received $7.40 an hour, but, at the time, Connecticut state minimum wage was set at $8.70. From May 2012 to May 2014, Zelepos reportedly failed to pay employees properly for their overtime work, paid employees in cash, and worked employees more than 93 hours per week. NBC reports that Zelepos is being ordered by the DOL to pay a total of $105,165.94 in back pay as well as $23,400 in civil penalties. Overtime: Officials say some employees worked 93 hours per week and Zelepos failed to properly pay and document their overtime hours. Zelepos is providing the DOL with payroll checks of the 110 employees ranging from $30 to more than $15,000, according to NBC. Gary K. Pechie, Wage and Workplace Standards director, said in a statement that similar situations occur 'far too often.' Fired: NBC reports that the Connecticut Department of Labor is now investigating a claim that one employee was fired after trying to report wage violations to the DOL. Pechi stated: 'Every employee deserves to be paid the required amount for the work that they do, and this is a flagrant example of an employer taking advantage of their workforce. 'We encourage any individual who believes they have not been paid properly for hours worked to file a complaint with the Labor Department.' NBC reports that the DOL is now investigating a claim that one employee was fired after trying to report wage violations to the DOL. Mystic Pizza, located in Connecticut, has been open since 1973, but became popularized by the 1988 film 'Mystic Pizza' starring Julia Roberts and Annabeth Gish. The film was named after the Connecticut restaurant caught the eye of the film's screenwriter, Amy Holden Jones | Summary: John Zelepos is being ordered by the Department of Labor to pay a total of $105,000 in back pay to 110 Mystic Pizza employees. Zelepos worked some employees more than 90 hours a week. He owes more than $23,000 in civil penalties for 155 minimum wage and overtime violations. One employee was reportedly fired for reporting wage violations to the Connecticut Department of Labor. | 603 | 102 | cnn_dailymail | en |
Summarize: FIELD OF THE INVENTION The present invention relates to an applicator set for a powdered or paste product, in particular a cosmetic product, of the type including a container or bottle provided with a neck and intended to contain the product, a cap to close the container, and an applicator disposed at the end of a rod attached to the cap. The invention relates more particularly, but not exclusively, to a set for applying mascara or eyeshadow. BACKGROUND OF THE INVENTION When the container of such a set contains a powder, such as a powder used in makeup, without an oily binder, it is generally possible to obtain good distribution of the powder without difficulty on the applicator mounted at the end of the rod; this applicator then makes it possible to apply the powder relatively satisfactorily. Nevertheless, such a powder does not adhere well to the surface, such as a eyelid, because the surface is too "dry". Hence it is preferable to use a powder including an oily binder, which better assures the adherence of the powder to the surface to which it is applied. Nevertheless, even though such a powder may be slightly oily, if it is in the form of loose powder it is more difficult to distribute with an applicator set of the type described above, in which the applicator disposed on the end of the rod in general has a tapered shape, such as a frustoconical shape. Such a powder including a binder, which will be called "oily loose powder" herein, tends to form clumps, which makes it difficult if not impossible to saturate the applicator. Moreover, the oily loose powder itself spreads poorly over the applicator. In French Patent Application 87 12073, filed on Aug. 31, 1987, an applicator set of the above type has been proposed in which the bottle has means, at its end opposite that provided with a cap and the applicator, on which the user can act in order to compact the product around the applicator, and elastic means for returning the compacting means to the position of repose when the user ceases such action. Preferably, the compacting means include a piston mounted to slide in the bottle and connected to a pushbutton that projects beyond the end of the bottle opposite the cap. Nevertheless, when one presses on the pushbutton to compact the product around the applicator, the air contained in the reservoir is compressed. Under these conditions, a "piston effect" takes place inside the container, and consequently the compacted product does not cling in a favorable manner to the applicator. OBJECT AND SUMMARY OF THE INVENTION The present invention makes it possible to overcome this disadvantage, and to this end proposes allowing the air to escape to the outside at the moment of compacting, via the applicator carrier tube; the applicator itself is made of a porous and spongy material, such as an open-cell foam, in such a way that the compressed air can pass through it before being evacuated. Additionally, the present invention makes it possible to apply a cream as well, and the compacting and passage of air through the applicator permits better filling and saturation of the applicator. Hence the subject of the present invention is the novel industrial product comprises an applicator set for a powdered or paste product, in particular a cosmetic product, including a container provided with a neck and intended to contain the product, a cap for closing the container, and an applicator disposed at the end of a rod attached to the cap. The container includes means for compacting the product around the applicator means on which the user can act to generate a compacting of the product, and elastic means to return the compacting means to their position of repose when the user ceases the compacting action; in accordance with the invention, the applicator is made of a material permeable to air, and the rod of the applicator includes at least one passage for evacuation of the air which is present in the container and which is compressed at the time of compacting; the air to be evacuated penetrates the rod after passing through the material permeable to the air of which the applicator is made, and escapes via the evacuation passage or passages to outside the cap. In accordance with a preferred embodiment of the applicator set according to the invention, the material of the applicator is a porous and spongy material. The rod advantageously comprises a hollow element defining an axial conduit for passage of the air to be evacuated, and the rod includes a terminal portion that supports the applicator; there are lateral air passage conduits in the terminal portion that discharge into the axial conduit. In accordance with another feature of the present invention, at the opposite end of the applicator the rod terminates in a tip assuring the solid attachment of the rod and cap; the air evacuation passage or passages communicate with the outside via at least one conduit made between the cap and the rod. In particular, this tip may be introduced with force into the cap; the lateral walls facing the tip and the cap include radial fins, between which the air evacuation conduits are made, and the free end of the tip is spaced apart from the base of the cap. Moreover, the fins carried by the cap assure the interlocking of the cap onto the end of the neck of the container when the set is in the closing position. In the case where the compacting means include a piston mounted to slide in the container, this piston being connected to an external pushbutton, it may be advantageously provided that the neck comprises a tubular element mounted to slide in an opening made in the upper wall of the container, and at its end adjacent to the container it has an external collar comprising the piston and returned to inside the aforementioned wall of the bottle by the action of the elastic return means. The means for controlling the piston comprise the cap, put in place on the container in its position of closure of the container, the distance between the free edge of the cap and the upper wall of the container in the aforementioned position of closure being equal at least to the maximum travel of the piston. The elastic return means may include a helical spring disposed between the piston and the base of the container or an element carried by the base of the container. The piston advantageously comprises a squeegee holder, and to this end includes a lip for squeegeeing the applicator. The container may comprise a tubular body bent at one of its ends to comprise the upper wall of the bottle traversed by the neck, the body being closed at its other end with a removable stopper. It may also be provided that the compacting means comprise a piston connected to an external pushbutton which projects beyond the end of the container opposite the neck. Preferably, the piston is then provided with a central hole making it possible to fill the applicator and with a stopper device to close this central hole after filling of the applicator. Preferably, the elastic return means comprise a helical spring disposed between the piston and an element that partially surrounds the applicator. The element surrounding the applicator is preferably attached to a part that forms a neck having a thread, mounted with force in the container. When the product to be applied is a powder, the powder includes microcapsules including liquid substances such as oils, binders or active ingredients, such as moisturizing agents, or substances for hypoallergenic, anti-wrinkle or dermatological treatment; these microcapsules release their contents in the vicinity of the applicator at the time of the compacting phase. For better comprehension of the subject of the present invention, two exemplary embodiments will be described in detail below, referring to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWING FIGS. 1 and 2 are axial sections of the same applicator set according to the invention, with the cap in place on the container and the powder in the loose state in FIG. 1 and in the state in which it has been compacted about the applicator in FIG. 2; FIG. 3 is a longitudinal section through another applicator set according to the invention, with the cap in place on the container. DESCRIPTION OF THE PREFERRED EMBODIMENTS Turning first to FIGS. 1 and 2, an applicator set 101 is shown for a powdered product P, in particular a cosmetic product, such as a mascara mixed with the oily binder and comprising an oily loose powder. The container of the applicator set 101 is a bottle 102 intended to contain the product P. The bottle 102 has the general shape of a cylinder of revolution; it is provided with a neck 103 arranged to slide axially in the bottle 102 as will be described hereinafter. A removable cap 104 is intended to close the bottle 102 being adapted to the free end of the neck 103. The cap 104 comprises a brush making it possible to manipulate an applicator 106 carried by the end of a rod 107 attached to the cap 104 at its tip 105 opposite the applicator 106. The applicator 106 penetrates to the interior of the bottle 102 via an orifice 109 bounded by a squeegee lip 120, the function of which is to exert a squeegee action on the applicator 106, to eliminate excess makeup product picked up by the applicator 106 inside the bottle 102. The bottle 102 includes a cylindrical body 136, which is bent at a right angle toward the inside at one of its ends, to make annular wall 137 the inner edge of which defines a circular opening 138. In the vicinity of the end opposite the wall 137, the body 136 has a thread 139 on its inside, intended to cooperate with the external thread 140 of the cylindrical flange 141 of a removable stopper 142 intended to plug the body 134 at that end. The stopper 142 includes a flat bottom 143, having a diameter corresponding to the outside diameter of the body 136, and the aforementioned flange 141 is connected to this flat bottom. The outer edge 143a of the bottom 143 is rounded. In the vicinity of its free edge, the flange 141 includes a recess on the inside of its inner wall, forming an annular bearing 144 on which the end of a helical spring 129 is supported; this spring comprises the elastic return means R for the means for compacting the powder P about the applicator 106, as will be described hereinafter. When the stopper 142 is screwed onto the body 136, a reservoir 111 is formed in the body 136, intended to contain the product P. The neck 103 comprises a very elongated cylindrical tubular element, the outside diameter of which is slightly less than the diameter of the circular opening 138. On its free end, the inner edge of the neck 103 is beveled, as a result of which the applicator 106 is easy to introduce into the bottle 102 because of the frustoconical flaring out of the opening 114 thus effected. At the opposite end, the neck 103 is folded at a right angle toward the outside along an annular collar 124, which forms a piston comprising the compacting means C. The element 124, which is intended to be placed inside the bottle 102 by being pressed by the spring 129 against the annular wall 137, has a total diameter substantially equal to or very slightly less than the inside diameter of the body 136; it has a cylindrical flange 145 that is coaxial with the neck 103, and opposite the neck this flange has the same outside diameter as the element 124. On its entire inside edge, the element 124 also includes a bead oriented toward the inside and comprising the aforementioned squeegee lip 120, defining the orifice 109, and also includes a short flange 146 which is coaxial with the flange 145 and is oriented toward the same side where the inner wall is flared, comprising the extension of the squeegee lip 120. The zone of the annular wall 147 of the element 124 between the two flanges 145 and 146 comprises a support bearing for the other end of the helical spring 129, which in the assembled position of the set extends axially within the reservoir 111. The cap comprises a peripheral flange 148 connected to a base 149; the base 149 includes three radial fins 150, which are distributed at regular intervals on the periphery of the base and the function of which will be described hereinafter. Moreover, between its free edge and the lower edge of the radial fins 150, the inner wall of the flange 148 includes longitudinal fins 151, distributed at regular intervals. The rod 107 is a hollow elongated element having a main cylindrical portion 152 intended to be accommodated inside the neck 103 and having an outside diameter that is smaller than the inside diameter of this neck 103. This main portion 152 is retracted at one end along a frustoconical wall 153, so that it ends in a tapered point 154 through which a narrow axial conduit passes comprising the elongation of the interior space of the main portion 152. This tapered point 154 is in turn penetrated by a plurality of transverse conduits 155 discharging into the aforementioned axial conduit. The tapered terminal portion 154 also comprises the support surface for the applicator 106, which is made from a porous foam having open cells. In the assembled position, the applicator 106 is accommodated entirely inside the reservoir 111, with the transition zone between the wall 153 and the tapered point 154 being located in alignment with the squeegee lips 120. Moreover, the diameter of the applicator 106 in its largest portion is notably greater than the diameter of the circular orifice 109, such that the squeegee lips 120 can exert their squeegee action on the applicator 106 when the applicator is removed from the reservoir 111. Remote from the point 154, the main portion 152 of the rod 107 flares along a frustoconical portion 156 to form the aforementioned tip 105. This tip 105 comprises the portion assuring the attachment of the rod 107 to the cap 104. To this end, the tip 105, comprising a cylindrical tubular element, has fins 157 on the outside, which are intended to be placed between the fins 151 of the cap 104, thus assuring a pressed fit of the rod -07 in the cap 104. However, in the assembled position, the fins 151 and 157 do not abut, thus forming longitudinal air outlet conduits along the flange 148 of the cap 104, which communicate with the central portion of the rod 107 via the passages left open between the fins 150. In the assembled position of the cap 104, the frustoconical wall 156 presses against the edge of the neck 103 in the opening 114, thus assuring the tightness of the applicator set 101. The assembly of the applicator set according to the invention is effected as follows: The rod 107 carrying the applicator 106 is attached with its tip 105 in the cap 104 until the tip 105 abuts against the radial fins 150; the retention of these two elements is assured by the abutment of the edges of the longitudinal fins 151 and 157 against the lateral walls of the tip 105 and flange 148, respectively, of the cap 104. In this position, the frustoconical connection zone 156 and the end zone of the main portion 152 of the rod 107 near the connection zone 156 are contained within the cap 104. The element comprising the neck 103, ending in the element 124, is introduced axially into the body of the bottle 136, by the end carrying the thread 139, until the element 124 abuts against the annular wall 137 of the bottle 102. The free end of the neck 103 is topped with the cap 104, and the fins 151 come to rest at their end against the neck 103, in such a way as to assure the closing engagement of the cap 104 with a sufficient locking action, with the neck 103 resting against the frustoconical wall zone 156 of the rod 107. The partial set thus made is then retracted, and the spring 129 is then put in place, inside the reservoir 111 which then opens at the top; then the reservoir 111 is filled with the powder P, and the stopper 142 is screwed on. The set is now ready for use. When the user wishes to apply the powder P to a surface, for example to the eyelids, the user may begin by pressing on the cap 104 in the direction of the arrows f2 shown in FIG. 2. In that case, the cap 104 functions like a pushbutton, causing the displacement of the element 124, which functions as the compacting piston, and the powder P is then compacted around the applicator 106, which undergoes the same axial translation, since it is attached to the cap 104. In this compacting operation, the air located in the reservoir 111 escapes through the pores of the foam comprising the applicator 106 and then through the transverse conduits 155 and the central conduit of the point 154, then via the axial conduit of the rod 107, to escape via the conduits formed along the cap 104. This escape of air toward the outside is symbolically represented by the arrows f in FIG. 2. The height of the neck 103 measured between the upper wall 137 of the bottle 102 and lower edge of the flange 148 of the cap 104, in the position of repose of the set (FIG. 1) is greater than or equal to the maximum distance that the piston 124 can travel. In this way, the air can always escape via the edge of the cap 104. The displacement of the piston 124 causes an even compacting of the powder P about the applicator 106, with t he effect of filling this applicator 106 with powder. The user then releases the pressure on the cap 104, and the spring 129 pushes the piston 124 back, thus placing the powder P in suspension. The applicator 106 is then ready for use. The user can remove the cap 104 with the applicator 106, suitably filled with powder for use. It can also be noted that filling of the device can be done via the neck 103 after the stopper 142 is screwed on. Also, the device enables refilling of the reservoir 111 when it has been emptied of powder that it initially contained; it suffices to unscrew the stopper 142 to perform refilling again, or this can also be done via the neck 103 FIG. 3 shows a different applicator according to the invention, in which the compacting means is a piston mounted at the base of the container, and the cap is screwed onto the neck of the container. In FIG. 3, the applicator set is identified by reference numeral 201. It includes a container in the form of cylinder of revolution 202, closed by a part forming a neck 203, and also includes a cap 204 carrying an applicator 206 at the end of a rod 207. On the end of the container 202 opposite the neck 203, there is a piston 205 that cooperates with a helical spring 209. The cylindrical container 202 includes a cylindrical lateral wall 221, and on the end opposite the neck includes an annular collar 222 which is perpendicular to the lateral wall 221. The collar 222 forms a circular opening 223. The piston 205 is made up of two cylindrical parts: one, 25-, has a cross section the outside diameter of which is slightly less than the diameter of the opening 223, and the other part, 252, has cross section the outside diameter of which is greater than the former, but slightly smaller in cross section than the inside diameter of the cylindrical wall 221 of the container 202. The two cylindrical parts are joined via a perpendicular annular portion 253. The piston is closed with lid 254 which is affixed by telescoping, with the aid of two concentric flanges 254a and 254b on the outside end of the cylindrical part 251 of the piston 205. The cylindrical part 251 of the piston projects through the circular opening 223 made at the end of the container 202 and serves as a pushbutton to actuate the piston. The piston is attached such that it can reciprocate and rotate in the container 202. The cylindrical helical spring 209 is supported on one side on the annular portion 253 of the piston 205 and on the other on the part forming the neck 203; it extends axially in the container 202. The coils of the spring 209 are disposed in the vicinity of the lateral wall 221 of the container 202; taken as a whole, the spring has a diameter such that it can surround the applicator 206 when the set 201 is closed. The part forming the neck 203 is provided on the inside with an axial cylindrical opening 231. At one of its ends, the outer wall includes a cylindrical annular portion 232 having a diameter slightly less than the inside diameter of the lateral walls 221 of the container 202; an outer collar 233 and a cylindrical portion 234 the outside diameter of which is smaller than the cylindrical portion 232; the cylindrical portion 234 is provided with an external thread 235 at one end. The cylindrical portion 232 is introduced with force into the container 202, with the collar 233 serving as a stop and assuring tightness. The spring 209 is surrounded by the cylindrical portion 232. On its outside, the cap 204 has the shape of a cylinder of the same diameter as the container 202 and is provided with a base 241. The inside wall of the cap 204 on the side of the base 241 thereof is provided with radial fins 243, and the side of its open edge with an internal thread 242 corresponding to the external thread 235 of the part forming the neck. The radial fins 243 have a length such that they are located entirely beyond the part forming the neck 203 when the applicator set 201 is closed. The thread carried on the neck 203 includes fine axial grooves. The applicator 206 and the rod 207 are the same shape as the applicator 106 and rod 107 shown in FIGS. 1 and 2. The applicator set 201 shown in FIG. 3 functions as follows: When the user wishes to apply the powder P to a surface, for example to the eyelids, the user begins by pressing on the part 251 forming a push element for the piston as indicated by the arrow F. The piston 205 is displaced axially in the container 202, compressing the helical spring 209 and compacting the powder about the applicator 206. In this operation, the air that is contained in the container 202 escapes through the pores of the foam comprising the applicator 206, then via the axial conduit of the rod 207 and then via the space made between the rod 207 and the cap 204 by the fins 243 and finally via the grooves of the thread of the neck 203 and the space included between the part forming the neck 203 and the cap 204. This escape of air is represented by the arrows f in FIG. 3. The compacting and the passage of the air have the effect of filling the applicator 206 with powder. The user then relaxes the pressure on the part 251 of the piston acting as a pushbutton, and the spring pushes the piston 205 back until the annular portion 253 of the piston 205 abuts the collar 222 of the container 202. The applicator 206 is then ready for use, and the user can unscrew the cap 204, remove the applicator 206, now suitably filled with powder, from the container 202 and proceed to apply the powder. It should be noted also that refilling of the container 202 can be done simply by opening the lid 254 of the piston 205 and pouring the powder P in through the opening thus formed. It will be understood that the embodiments described above and shown in the drawings are purely given as examples and may undergo any desired modification without departing from the scope of the invention. | Summary: The applicator set (101) includes a bottle (102), provided with a neck (103) and intended to contain a product (P), a cap (104) for closing the bottle, and an applicator (106) disposed at the end of a rod (107) attached to the cap (104). The bottle (102) includes means (C) for compacting the product around the applicator (106), upon which means the user can act, and elastic means (R) for returning the compacting means (C) to the position of repose. The applicator (106) is made of a porous material, and the air present in the bottle (102), which is compressed at the time of compacting, can escape via the cap (104), passing through the hollow rod of the applicator (107), after having passed through the porous material of the applicator (106). | 5,963 | 216 | big_patent | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``National Education Advancement (NEA) and Teacher Relief Act''. SEC. 2. CREDIT FOR CONTRIBUTIONS FOR THE BENEFIT OF ELEMENTARY AND SECONDARY SCHOOLS. (a) In General.--Subpart B of part IV of subchapter A of chapter 1 of the Internal Revenue Code of 1986 is amended by adding at the end the following new section: ``SEC. 30B. CREDIT FOR CONTRIBUTIONS FOR THE BENEFIT OF ELEMENTARY AND SECONDARY SCHOOLS. ``(a) Allowance of Credit.--There shall be allowed as a credit against the tax imposed by this chapter for the taxable year an amount equal to 75 percent of the qualified charitable contributions of the taxpayer for the taxable year. ``(b) Maximum Credit.-- ``(1) Individuals.--In the case of a taxpayer other than a corporation, the credit allowed by subsection (a) for any taxable year shall not exceed $500 ($1,000 in the case of a joint return). ``(2) Corporations.--In the case of a corporation, the credit allowed by subsection (a) shall not exceed $100,000. ``(c) Qualified Charitable Contribution.--For purposes of this section-- ``(1) In general.--The term `qualified charitable contribution' means, with respect to any taxable year, the aggregate amount allowable as a deduction under section 170 (determined without regard to subsection (d)(1)) for cash contributions-- ``(A) to a school tuition organization, ``(B) for the improvement, renovation, or construction of a school facility that is used primarily to provide education at the elementary or secondary level, and ``(C) for the acquisition of computer technology or equipment (as defined in subparagraph (E)(i) of section 170(e)(6)), or for training related to the use of such technology or equipment, for use in a school facility described in subparagraph (B). ``(2) Certain expenses of elementary and secondary school teachers.-- ``(A) In general.--In the case of an individual who is an eligible educator, the term `qualified charitable contribution' includes amounts allowable as a deduction by section 162 paid or incurred by the eligible educator in connection with books, supplies (other than nonathletic supplies for courses of instruction in health or physical education), computer equipment (including related software and services) and other equipment, and supplementary materials used by the eligible educator in the classroom. ``(B) Coordination with exclusions.--An amount shall be allowed as a credit under this section for expenses described in subparagraph (A) only to the extent the amount of such expenses exceeds the amount excludable under section 135, 529(c)(1), or 530(d)(2) for the taxable year. ``(3) School tuition organization.-- ``(A) In general.--The term `school tuition organization' means any organization which-- ``(i) is described in section 170(c)(2), ``(ii) allocates at least 90 percent of its gross income and contributions and gifts to elementary and secondary school scholarships, and ``(iii) awards scholarships to any student who is eligible for free or reduced cost lunch under the school program established under the Richard B. Russell National School Lunch Act. ``(B) Elementary and secondary school scholarship.--The term `elementary and secondary school scholarship' means any scholarship excludable from gross income under section 117 for expenses related to education at or below the 12th grade level. ``(4) Eligible educator.-- ``(A) In general.--The term `eligible educator' means, with respect to any taxable year, an individual who is a kindergarten through grade 12 teacher, instructor, counselor, principal, or aide in a school for at least 900 hours during a school year. ``(B) School.--For purposes of subparagraph (A), the term `school' means any school which provides elementary education or secondary education (kindergarten through grade 12), as determined under State law. ``(5) School facility.--The term `school facility' shall not include any stadium or other facility primarily used for athletic contests or exhibitions or other events for which admission is charged to the general public. ``(d) Special Rules.-- ``(1) Denial of double benefit.--Amounts taken into account under subsection (a) shall not be taken into account in determining any deduction allowed under section 162 or 170. ``(2) Application with other credits.--The credit allowable under subsection (a) for any taxable year shall not exceed the excess (if any) of-- ``(A) the regular tax for the taxable year, reduced by the sum of the credits allowable under subpart A and the preceding sections of this subpart, over ``(B) the tentative minimum tax for the taxable year. ``(e) Election to Have Credit not Apply.--A taxpayer may elect to have this section not apply for any taxable year.''. (b) Clerical Amendment.--The table of sections for subpart B of part IV of subchapter A of chapter 1 of such Code is amended by adding at the end the following new item: ``Sec. 30B. Credit for contributions for the benefit of elementary and secondary schools.''. (c) Effective Date.--The amendments made by this section shall apply to taxable years beginning after December 31, 2003. SEC. 3. REVISION OF DEFINITION OF SCHOOL FOR PURPOSES OF QUALIFIED ELEMENTARY AND SECONDARY EDUCATION EXPENSES. (a) In General.--Paragraph (4) of section 530(b) of the Internal Revenue Code of 1986 (defining qualified elementary and secondary education expenses) is amended-- (1) in clauses (i) and (ii) of subparagraph (A), by striking ``public, private, or religious'', and (2) in subparagraph (B), by inserting after ``any school'' the following: ``, including a public, private, religious, or home school,''. (b) Effective Date.--The amendments made by this section shall apply to taxable years beginning after December 31, 2003. | Title: To amend the Internal Revenue Code of 1986 to allow a credit against income tax for teacher classroom supply expenses, for improving elementary and secondary education, and for contributions for scholarships to attend elementary and secondary schools, and for other purposes Summary: National Education Advancement (NEA) and Teacher Relief Act - Amends the Internal Revenue Code to allow a tax credit for 75 percent of charitable contributions made by a taxpayer: (1) to a school tuition organization that provides certain scholarship aid; (2) for the improvement, renovation, or construction of an elementary or secondary school facility; and (3) for the acquisition of computer technology or equipment, or for related training, for use in an elementary or secondary school facility. Limits the dollar amount of such credit to $500 for individuals and $100,000 for corporations. Allows elementary or secondary school teachers, instructors, counselors, principals, or aides who work at least 900 hours during a school year to claim the tax credit provided by this Act for their expenses in connection with books, supplies (other than nonathletic supplies for courses of instruction in health or physical education), computer equipment (including related software and services), and other equipment and supplementary materials used in the classroom. Revises the definition of "school" for purposes of defining qualified elementary and secondary education expenses to include public, private, religious or home schools. | 1,525 | 299 | billsum | en |
Write a title and summarize: Colonies of bacterial cells can display complex collective dynamics, frequently culminating in the formation of biofilms and other ordered super-structures. Recent studies suggest that to cope with local environmental challenges, bacterial cells can actively seek out small chambers or cavities and assemble there, engaging in quorum sensing behavior. By using a novel microfluidic device, we showed that within chambers of distinct shapes and sizes allowing continuous cell escape, bacterial colonies can gradually self-organize. The directions of orientation of cells, their growth, and collective motion are mutually correlated and dictated by the chamber walls and locations of chamber exits. The ultimate highly organized steady state is conducive to a more-organized escape of cells from the chambers and increased access of nutrients into and evacuation of waste out of the colonies. Using a computational model, we suggest that the lengths of the cells might be optimized to maximize self-organization while minimizing the potential for stampede-like exit blockage. The self-organization described here may be crucial for the early stage of the organization of high-density bacterial colonies populating small, physically confined growth niches. It suggests that this phenomenon can play a critical role in bacterial biofilm initiation and development of other complex multicellular bacterial super-structures, including those implicated in infectious diseases. The past few decades witnessed an emergence of the realization that bacterial cells in their natural environments are not asocial, but can exist as colonies with complex organization and exhibit sophisticated and highly regulated collective behaviors [1–5]. Consequently, significant efforts have been made to investigate the collective behavior of bacteria in various settings, with a particular emphasis on the formation of highly organized, multicellular super-structures. Instances of such colony formation include tightly packed bacterial “pods” in epithelial cells, colonies of luminescent bacteria in light organs of marine animals, or biofilms forming on plastic or glass surfaces in various high-humidity environments [6–10]. One important aspect of these naturally occurring tightly packed bacterial colonies (henceforth referred generically to as biofilms) is that they frequently arise despite, and possibly in response to, unfavorable environmental conditions including various types of chemical stress, variable temperature, fluid flow, the host immune system, and limited supply of nutrients [5]. In the initial stages of the biofilm development, it is crucial for bacterial cells to overcome the above-mentioned adverse environmental conditions, while laying foundations for highly ordered, mature biofilm structures. Recent studies have revealed that one of the important initial steps in this process might be for bacterial cells to actively seek out small cavities and populate them, reaching very high densities [11–13]. In addition to providing partial shelter, physical confinement might facilitate the onset of quorum sensing that is thought to be important for the successful progression of biofilm development. However, there are also severe potential disadvantages to forming packed colonies that are partially isolated from the surrounding environment, including increasingly poor nutrient supply and waste removal, as well as the possibility of disorganized expansion leading to cell damage and even blockage of cell escape from the growth niches. How cells cope with these constraints to successfully initiate biofilm development is currently unclear. A clue to understanding cell behavior in these early stages of biofilm development might come from the high degree of multicellular organization found in stalk formation of yeast cells emerging from microscopic pits in agar gels [14,15]. Initial confinement of cells to small cavities and mechanical interaction between cells and the cavity walls appeared to be essential not only for the formation of complex tall structures uncharacteristic of lab yeast strains, but also for the high degree of functional order and differentiation within these structures. Colony organizations in various organisms such as Bacillus subtilis and Escherichia coli, that were visualized using scanning and transmission electron microscopy [2], also show strikingly well-ordered multicellular arrays within the colonies. Recently, with the aid of advanced microscopy systems, biofilm structures were also found to comprise millions of bacterial cells in regular arrangements that facilitate the physiological interactions within the community [9]. It is of interest therefore to investigate whether this functional organization in biofilms, as in yeast stalks, might emerge from the initial ordering of bacterial colonies growing in small cavities—ordering that might facilitate nutrient exchange and relieve the mechanical stress stemming from cell proliferation. Although these dynamical reorganizations of the biofilm-like bacterial colonies are likely to be controlled by cell–cell interactions, lack of convenient experimental platforms allowing a dynamic analysis of large, tightly packed colonies on the single cell level has hampered research progress in this area. Recently, we developed a microfluidic device with chemostatic microchambers, which allows bacterial cells to grow over multiple generations in controlled microenvironments [16]. However, these chambers were relatively deep (∼6 μm), making it difficult to reach single-cell resolution at high cell densities. In addition, the device was designed to prevent the escape of cells from the chambers, and the packed colonies could only be monitored over a very limited number of cell generations. In this study, we sought to overcome these limitations and create a device for monitoring tightly packed colonies of actively dividing cells in chambers with different geometries over at least 24 h with single-cell resolution. Using a combination of modeling and experiments in these new devices, we found that developing E. coli colonies achieve progressively higher levels of spatial organization, enabling them to increase the nutrient supply and the efficiency of escape from the chamber. The results of the study suggest added importance of the asymmetrical shape of some bacterial species and have direct implications for biofilm organization. The device we describe can also have wider applications for robust long-term culture of bacterial cells under controlled conditions with real-time microscopy at single-cell resolution. Similar to the previously described microfluidic chemostat [16], the main functional area of the microfludic device (Figure 1) is an array of flow-through channels and chambers between the channels. The symmetric binary branching of the flow-through channels from a single channel on both the inlet and outlet sides leads to highly balanced pressures at opposite sides of the chambers. Because the depth of the chambers is much smaller than the depth of the flow-through channels (∼1. 5 μm versus 15 μm; Figure 1A), the chambers are much more resistant to flow than the channels. Therefore, there is practically no active flow through the chambers (flow at 0. 1–0. 2 μm/s for a typical channel flow rate of ∼100 μm/s), and the exchange of chemicals between them and the flow-through channels occurs by diffusion only. There are three important differences between the device in Figure 1 and that described in [16]. First, the depth of the chambers of our device is close to the diameter of E. coli cells (∼1 μm) and substantially smaller than the cell length (∼3 μm). Therefore, all cells in the chambers are oriented in the plane of the device and create a monolayer, making it possible to detect cellular responses at a single-cell resolution. Second, there are no capillaries impermeable for cells between the flow-through channels and the chambers. Therefore, there are no physical barriers for cells to enter and exit the chambers. Third, although the distance between all flow-through channels is the same (140 μm), the chambers greatly vary in shapes (Figure 1B). The shapes of the outer boundaries of the chambers include circles, squares, diamonds, triangles, and strips. Furthermore, many chambers have one or several posts inside, adding inner boundaries to the geometries (Figure 1B). The posts have different sizes and are shaped as circles, squares, diamonds, triangles, parallel rectangles, and crosses. As in the cells in a microfluidic chemostat [16], the E. coli cells loaded into the chambers were found to readily form microcolonies. The microcolonies started from as few as 1–2 cells and eventually filled the chambers completely, with cells being in a densely packed state. However, since cells could freely exit the chambers into the adjacent flow-through channels, cell proliferation was not limited in time and continued for as long as the flow of fresh medium in the flow-through channels was maintained. The combination of cell proliferation and escape led to a continuous collective motion of cells of the densely packed colonies toward the microchamber exits. The rate of flux of cells through the exits is proportional to the rate of colony expansion and, given constant volume of the chamber and constant number of cells in the colony in a steady state, the rate of flux is also proportional to the mean cell growth rate in the colony. We did not observe any noticeable decrease in the growth rates of high-density colonies during prolonged continuous incubation, as judged by the characteristic rate of motion of cells toward chamber exits (see, e. g., Figure S3A). For effective visualization of the organization of microcolonies, we used a strain of E. coli, transformed with a low-copy plasmid carrying green fluorescent protein (GFP) controlled by the truncated version of the Vibrio fischeri lux operon responsive to exogenously added V. fischeri–specific autoinducer [17], N-3-oxo-hexanoyl homoserine lactone (HSL), but deficient in endogenous AI production (Figure S1). In the presence of exogenously added autoinducer supplied with the medium ([AI] = 10 nM), the GFP mediated fluorescence allowed us to visually identify individual cells and perform analysis of their shape, size, and orientation. We analyzed images of the colonies in different chambers (see Videos S1–S8 for typical examples) and were surprised to observe that, at high densities, the orientations of cells were often anisotropic and highly correlated over distances much larger than the cell length. Typical evolution of a colony in one of the chambers is shown in Figure 2A. Both inner and outer boundaries of the chamber are shaped as rhombi (“rhombus in a rhombus” or RIR chamber). The cell growth initiated in region I, and the orientations of cells appeared random in the beginning. The microcolony then filled the entire chamber to dense cell packing in 8–9 h of growth. (Dense packing in a subcolony was defined as a state in which there was no cell-free region with the area equal to or greater than the area of a daughter cell following a cell division.) As the colony reached dense packing, most cells gradually became oriented along the direction of the collective cell flow toward the chamber exits, which frequently coincided with the directions of the internal and external walls of the chamber. To quantify the orientation of cells, we processed fluorescence images of the chambers using two different methods that we termed the “gradient analysis” and the “major-axis analysis” (Protocol S1 and Figure S2). In the major-axis analysis, the images were segmented to outline individual cells. Following this, the orientations of the major axes of the cells were determined. In the gradient analysis, cell orientation was estimated by determining the direction of the fluorescence gradients from the pixel intensity matrices, without explicit identification of individual cells. Sharp changes of fluorescence occur at cell boundaries, and the directions of fluorescence gradients are perpendicular to the orientation of cells. We applied both methods to sequences of images taken with an interval of 1 h in selected regions of the chambers and found that the two methods consistently yielded similar results (Figure S2). In region I of the RIR chamber (Figure 2A), at the early time points 0 h and 2 h, the orientation histograms had nearly uniform distributions, indicating that cells were oriented randomly (Figure 2B). As the cell density in the colony increased, the distribution of cell orientations evolved to a shape with a peak at 45°, the direction of the chamber walls and of the collective cell flow in this region. Histograms of the orientation of cells in densely packed states in the other selected regions of the chamber (Figure 2A) were similar to the distribution in region I (Figure 2C). In particular, all histograms had peaks at angles corresponding to directions of the chamber walls—135°, 135°, and 45° for regions II, III, and IV, respectively. The shapes of the orientation histograms of densely packed colonies at distinct time points differed relatively little (Figure 2C), and there were no detectable tendencies in the variation of their shapes with time (Figure 2B). This invariance of histograms indicated that the microcolony reached a steady state in terms of cell orientation. To assess the evolution of cell orientation, we introduced an order parameter, β, calculated as the fraction of cells oriented within ±45° of the angle at the peak of the eventual steady state histogram (e. g., at 0°−90° for region I). We plotted the time evolution of β for the four regions of the chamber (Figure 2D). The value of β is 0. 5 for randomly oriented cells and it is 1 when all cells are aligned in the preferred direction, which is parallel to the chamber walls. Values of β ∼ 0. 5 with large variations in time, suggesting basically random orientation of cells with large fluctuations, were typical at the early stages of colony development when cell densities were low. As time progressed and cell densities increased, variations of β became minimal, as expected for a microcolony at a steady state. In this steady-state regime, the values of β were 0. 85 on average, indicating a major bias in the orientation of cells toward the directions of the nearby chamber walls. (Calculation of the order parameter using a reduced range of angles around the preferred orientation resulted in smaller values of β at the steady state but did not change the shapes of the curves.) The large value of the order parameter was attained in spite of the fact that the distance between the walls (∼30 μm) was much larger than the cell diameter. The cells in the region I reached a highly ordered state earlier, and in the region II reached it later, than cells in other regions, in accordance with the relative timing of expansion of the colony into these regions. In addition to the RIR chamber, we used the same methods to analyze the cell orientation in chambers with other shapes. Two typical examples are described here. One chamber was shaped as a rhombus with no internal boundary (Figure 3A), and the other was shaped as a circle with a square internal boundary (Figure 3B). In both chambers, the walls were substantially further apart from each other than in the RIR chamber, which could be expected to lead to less order in the orientation of cells. Surprisingly, the analysis of images of densely packed colonies in both chambers showed that, in most regions, the orientation of cells was highly correlated over large distances. In a chosen region of interest (e. g., regions I, III, IV, and V in Figure 3A and 3B), the preferred orientation of cells usually coincided with the orientation of a nearby wall and always coincided with the direction of flow of cells toward chamber exits. In the central region of the rhombus-shaped chamber (region I and inset I in Figure 3A), close to 95% of cells were oriented within ±45° from the vertical axis, which was the direction of flow toward the chamber exits (Figure 3C and 3D). The orientations of cells in region I were highly correlated over a distance ∼20 times the cell length, in spite of the absence of chamber walls nearby. The orientation order was similarly high in most other regions of the chamber. Exceptions were the side corners (region II and inset II in Figure 3A), where cells appeared to be oriented nearly randomly, as indicated by a close-to-uniform distribution of angles (Figure 3C) and the order parameter β ≈ 0. 5 in the steady-state regime. In the “square within a circle” chamber, the distributions of cell orientations in regions IV and V reached the steady states rapidly, and the order parameter attained high values β > 0. 9 (Figure 3C and 3D). In region III, the steady state was reached considerably later than in regions IV and V, and the cell orientation was less anisotropic, as indicated by a lower value of the order parameter, β ≈ 0. 8 (Figure 3C and 3D). In contrast to regions III and V, in region IV, the direction of flow of cells toward the exit (the flow direction defined the preferred orientation of cells) was orthogonal to the orientation of a nearby chamber wall. As a result, the flow pattern in region IV was rather complicated, with two fluxes of cells (from the left and from the right in Figure 3B) merging into one and with an analogue of a separation line in the middle. The observation that a high degree of correlation in cell orientation occurred only at high cell densities, in the absence of active cell movement, suggested that the correlation originated from purely mechanical forces. Locally, these forces result from contact interactions of cells with each other and from the constraint of 2-D motion in the plane of the chamber. Globally, the cells are mechanically influenced by their collective motion toward chamber exits. The pattern of this motion depends on the position and orientation of the chamber walls and exits. To investigate if cell self-organization could be solely due to these contact interactions, we simulated the development of a colony in a 2-D region corresponding to the footprint of a chamber, with cells modeled by 2-D objects with shapes, lengths, and widths typical for E. coli cells. The model simulations were based on our previously reported analysis of yeast colony growth [11] with appropriate modifications accounting for differences in cell shape, for symmetric cell division, for the presence of boundaries, etc. (see Methods for more details). Cells were assumed to grow steadily along their major axes, to divide at even time intervals, and to have spring-like deformation potentials. Forces experienced by cells were due to cell–cell and cell–wall interactions. We found that this simple mechanical model reproduced all the major features of colony expansion in different geometries, including the gradual onset of anisotropic orientation of cells with long-range spatial correlations at high cell densities. Both the time evolution and the steady-state distributions of the cell orientation angles in the RIR chamber strongly resembled the corresponding experimental results (Figures 2 and 4A). Similar evolution and steady-state patterns were obtained in the other modeled geometries (Videos S9–S11 and unpublished data). The agreement between the model and experiments corroborated the suggestion that the self-organization of cells within the colonies originated from purely mechanical effects. Since a preferred direction of orientation can only exist for nonspherical, elongated objects, the self-organization of E. coli cells in the colony and the anisotropy in their orientation are closely related to the elongated shape of E. coli cells. We used the model to examine how the orientation anisotropy and β in the high-density steady states depend on the ratio between the length and width of cells and simulated colonies composed of cells with maximum lengths shorter (0. 5L) and longer (2L, 4L) than “normal” (the normal length, L, corresponded to the average aspect ratio 3. 75: 1 calculated given that the cell lengths vary from half-maximal to maximal in the course of cell growth). We found that cells with the reduced aspect ratio had smaller orientation order and lower β than the “normal” cells (0. 81 vs. 0. 99 for region I). On the other hand, we found no significant difference in the anisotropy of orientation between the “normal” cells and the cells with the increased aspect ratios, with the β values of 1. 00 and 0. 97 for 2L and 4L cells versus 0. 99 for the normal cells. The results of the simulation suggest that the actual aspect ratio of E. coli (3: 1–4: 1) might be close to the minimal aspect ratio sufficient to ensure a high level of coordination of cell orientation within a colony. We further interrogated the model to evaluate the forces experienced by cells of different lengths at different stages of colony development (see Methods, Videos S12–S15). For cells of 0. 5L length simulated within a RIR chamber, the stress at the steady state increased with the distance from the chamber exits and was maximal along the horizontal axis of symmetry of the chamber (Figure 4C). The distribution of stress in the simulated colony was similar to the distribution of pressure in a model of a Newtonian fluid with spatially uniform source term, corresponding to a uniform and steady growth of cells in a chamber of the same geometry (Figure S3B). By contrast, for the longer cells (2L and 4L) than “normal” cells, the simulations indicated the existence of areas of high stress near the chamber exits. Near the exits, the directions of flow of cells in two merging streams change abruptly by 45°, the total width of the cell stream decreases, and the flow lines merge, causing many cells to become misaligned. The misalignment can lead to high stresses due to both growth of cells in the direction perpendicular to the flow and possible “stampede”-like exit blockage exacerbated by the convergence of flow lines (Videos S13 and S14). Additionally, we observed that if the cells were oriented perpendicularly to a nearby chamber wall and their growth was not directed toward one of the exits, the cells experienced high localized forces (Figure 4D, left and right corners of the chamber). This observation suggested that self-organization of colony expansion in parallel to the chamber walls may decrease the mechanical stress induced by cell growth. A high degree of anisotropy achieved in the steady-state colonies might affect the efficiency of diffusive transport of nutrients into and metabolites out of the bulk of the colony through the chamber exits. Indeed, a recent theoretical study suggested that increased anisotropy of a porous tissue can lead to a dramatic reduction of its tortuosity and enhancement of diffusion in a preferred direction [18]. The study also analyzed diffusion in a 2-D medium with an array of excluded regions having shapes of identical extended rectangles, oriented parallel to each other, which is a good approximation of the high-density steady-state colonies in the microchambers. The effective diffusion coefficient in the direction along the rectangles was found to be, where D is the diffusion coefficient of the medium without excluded regions, L1 is the longer, and L2 is the shorter axes of the rectangles. For E. coli cells with the aspect ratios 3: 1–4: 1, in a highly ordered steady state with β ≈ 1, one might thus expect an effective diffusion coefficient of 0. 75D to 0. 8D along the directions from the chamber' s exit into the bulk of the colony. For randomly oriented cells, Deff would drop to 0. 5D or even less, due to possible “dead-end pores”, i. e., blockages of openings between pairs of parallel cells by perpendicularly oriented cells. Thus, increasing the anisotropy of cell orientation within a colony is expected to enhance the supply of nutrients to internal regions of the colony and the evacuation of metabolites from the internal regions. To test this prediction, we took advantage of the sensitivity of the lux operon to glucose levels. Transcription in this operon is positively regulated by the cAMP-receptor protein (CRP) [19], which is progressively activated at decreasing glucose levels due to elevated intracellular cAMP concentrations. As a result, the level of GFP expression substantially increased at reduced glucose levels that were expected to be found in densely populated microfluidic chambers with impaired diffusive transport (Figure P3 of the Protocol S1). We found that, for all chamber shapes, fluorescence of individual cells increased as the colony became denser and filled the chamber, which was consistent with the expected reduction of glucose concentration. Moreover, in the high-density steady-state colonies, well-formed gradients of fluorescence were often observed, with cells becoming progressively less fluorescent toward the chamber exits (Figures 2 and 3 and Figure S5). Because the exits were a source of the exogenously added autoinducer, this type of distribution of fluorescence in the colony was inconsistent with the possibility that autoinducer did not reach cells in the internal regions. The variation of the glucose level appeared to be the only plausible explanation of this fluorescence distribution, and we concluded that the level of cell fluorescence could be used as an indicator of the local concentration of glucose. This conclusion was further corroborated by a high degree of correlation between the expression of GFP driven by the lux operon promoter and the expression of HcRed protein under exclusive control of cAMP-CRP complex (both expressed in the same strain of E. coli) in different cells growing in the same chamber [20] (Figure P4 of Protocol S1). We then investigated the evolution of GFP fluorescence in different regions of the previously analyzed chambers (Figure 5). In the case corresponding to region I in Figure 3A (rhombus-shaped chamber), we found that, following a maximum level reached around 13 h, the fluorescence intensity gradually dropped by approximately 30% (Figure 5A). Additionally, the fluorescence intensity gradient along a line connecting region I with a chamber exit gradually became shallower, indicating better penetration of the nutrients into internal regions of the colony (Figure 5A-ii). These observations could be explained by noting that the alignment of cells in the rhombus chamber reached its highest level at least 4 h after the colony became densely packed. Therefore, initially, nutrient transport through the colony would be hampered by decreasing intercellular spaces and increasing nutrient consumption leading to increased fluorescence intensity. However, over time, the transport efficiency can be improved by increased anisotropy within the colony, leading to a progressive decrease in the GFP signal at the colony interior. Consistent with this hypothesis, the drops in the GFP fluorescence intensity from the maximal levels were much more limited in the other two chambers analyzed, where by the time the colonies became densely packed, cells were already arranged in a highly anisotropic fashion (Figure 5B and 5C). Taken together, the results suggest that fluctuations in the nutrient supply available to the colony can depend on whether the colony self-organization accompanies or follows achieving the densely packed state. A significant increase in the colony growth rate, as judged by the rate of cell movement out of a chamber, which was observed in the rhombus but not RIR chamber at a later stage of experiment provided independent further support for this suggestion (Figure 5A-i and Figure S3A). This study has demonstrated that long-term growth of E. coli colonies within small enclosed spaces can be accompanied by self-organization of the colonies into states characterized by highly correlated cell orientation. A simple agent-based model incorporating the details of asymmetric cell shape, mechanical interactions arising from cell growth and division, and the confinement of colonies within specific boundaries has reproduced the salient features of this organization process. The success of the model strongly suggests that self-organization is due to localized mechanical cell–cell and cell–chamber wall interactions, and most importantly, elongated cell shape. When viewed on a coarse scale—that of hundreds of cells—the dynamics of steady-state colonies bear a marked similarity to the patterns of flow of Newtonian fluid continuously generated within confined spaces of various shapes. This similarity is exemplified by the coincidence of the streamlines of Newtonian fluid with the directions of the collective cell motion (Figure S3B). In addition, the areas corresponding to high fluid pressure overlap with those of increased stress experienced by cells with low aspect ratio (Figure S3B). These simple patterns are further refined on the scales comparable to the size of a single cell, where the effects of elongated shape of the cells become especially pronounced. In particular, it becomes evident that not only the direction of movement but also the orientations of individual cells become aligned with the predicted streamlines and also become mutually correlated. The directional correlation increases with increasing cell aspect ratio. This self-organization behavior, involving hundreds to thousands cells, contrasts with the previously reported transient local alignment of relatively small cell clusters ([21], Figure S4), and is strongly dependent on the geometry of the confining boundaries and chamber exits and on the direction of the collective cell flow (Figure 3D and Figure S5). Based on the results described here, we propose that colony self-organization can endow the constituent cells with at least two important advantages. First, orientation of cell growth toward the exits connecting microchambers to external space can lead to the relaxation of the stress experienced by the cells due to constriction by the chamber boundaries. In a cell population lacking such an orientational order, cell growth toward chamber walls can create local foci of high stress (see e. g., Figure 4D). Interestingly, increasing the cell aspect ratios beyond the values comparable to those of the wild-type E. coli does not seem to affect the degree of self-organization, but can increase the cellular stresses at the chamber exits due to rapid changes in cell orientation. In this regard, it is interesting to contrast the collective behavior of bacterial cells to the self-driven movement of a large crowd in a confined space. The model in [22] suggests that a combination of a relatively narrow bottleneck coupled with self-propelled, panic-like movement of people within a confined space can easily result in a blockage of the exits, leading to potentially widespread injuries. Arguably, bacterial cells in tightly packed colonies populating small cavities or other confining environments might face similar stampede-like blockage challenges. Our results suggest, however, that the potential for “bacterial stampedes” arises only for relatively long cells, which have feature sizes comparable to the dimension of the cavity exit openings. Therefore, from the standpoint of stress relief, one can speak of an optimal cell length, such that cells are long enough to undergo robust self-organization, and yet short enough to avoid increased stress at the exits from a confined space, and potential blockage of cell escape. It can be proposed that these constraints on cell shape, in addition to the potential importance of elongated cell shape for cell motility and other functional responses [23], might have contributed to the evolutionary pressure defining the aspect ratios of cells in many extant bacterial species, in particular those that have morphology similar to E. coli and form biofilms. The second advantage of anisotropic colony organization is the reduction of the tortuosity of the intercellular spaces progressively enhancing the diffusion of nutrients into the colony. Efficient diffusive transport of nutrients and metabolites is an important determinant of the well-being of bacterial cells within the bulk of the colony, ensuring sufficient energy supply for cell division and other functions. A corollary of this finding is that signal molecules might also diffuse more easily along the flow lines formed by the cells, thus introducing anisotropy into cell-cell communication, e. g., through quorum sensing. Our findings provide a fresh perspective on possible mechanisms of emergence of the structurally complex biofilms and other types of multicellular super-structures. Although the order inherent in biofilms is clearly shaped by a complex interplay of multiple processes, including intercellular signaling [24] and regulated migration of cell sub-populations within the developing and differentiating colonies [25], this order and the chances of survival of bacterial biofilms might critically depend on the initial self-organization of cells during their anchorage and expansion within small cavities and other niches in complex growth substrata. The complex mechanical interactions between cells themselves and between cells and physical boundaries confining them, determined in large part by cell morphologies, can help create patterns of extensive organization and enhanced chemical transport, which can lay the foundation of complex functional multicellular ensembles. The understanding of these processes might help control biofilm growth, and ultimately facilitate treatment of the related diseases. Finally, we note that the microchamber device presented here provides a convenient and effective way to observe bacterial cells over multiple generations and track the evolution of a colony at single-cell resolution. It enabled us to observe the individual cellular response in both fluorescence and phase-contrast microscopic settings. We envision that the flexibility of design and convenience of use will make this type of devices a platform of choice for many scientific and biotechnological applications. In our study, we defined the “orientation” of a cell as the angle that the cell forms with respect to the x-axis of the Cartesian coordinates of each image (inset in Figure 2B). Its range is [0°, 180°]. The custom software package to determine the cell orientations was developed and implemented in Matlab 7. 0. 0., including the image-processing toolbox. Time-lapse fluorescence images in the 16-bit TIFF format indexed by location and time stamps were used as the input. In the package, two different routines were applied to find the orientation of individual cells. (Gradient analysis and Major axis analysis; see Protocol S1 for a detailed description). Individual cells are explicitly modeled as separate agents and are assumed to have an area enclosed by two semi-circles attached to opposite sides of a rectangle of constant width but variable length. Positions and lengths of the cells are defined by two generalized coordinates designating the centers of the semi-circles attached to the rectangle. The dynamics of the colonies is considered to be dominated by viscous friction, with velocity rather than acceleration being proportional to the force. See Protocol S1 and Videos S17 and S18 for details of the model set-up and simulations. | Title: Self-Organization in High-Density Bacterial Colonies: Efficient Crowd Control Summary: Bacterial cells form colonies with complex organization (aka biofilms), particularly in response to hostile environmental conditions. Recent studies have shown that biofilm development occurs when bacterial cells seek out small cavities and populate them at high densities. However, bacteria in cavities may suffer from poor nutrient supply or waste removal, or disorganized expansion leading to blockage of cell escape. In this study, we observed Escherichia coli in a microfluidic device that allows direct observation of the growth and development of cell colonies in microchambers of different shapes and sizes through multiple generations. Combining this experimentation with computational analysis of colony growth and expansion, we characterize a process of colony self-organization that results in a high degree of correlation between the directions of cell orientation and growth of collective cell movement. We also find that this self-organization can significantly facilitate efficient escape of cells from the confines of cavities where they reside, while improving the access of nutrients into the colony interior. Finally, we suggest that the aspect ratio of the shape of E. coli and other similar bacteria might be generally subject to a constraint related to colony self-organization. | 7,723 | 272 | lay_plos | en |
Summarize: BACKGROUND OF THE INVENTION The invention pertains to fire fighting and particularly to a means for permitting a fire hose to lie slack while the fire truck unrolls the hose on the way to the site, and then permits the hydrant to deliver full pressure to the hose subsequent to arrival of the truck without requiring a fireman to remain at the hydrant to open the hydrant valve. Applicant has also filed two other applications, Ser. Nos. 636,556 now U.S. Pat. No. 4,022,421 and 722,709 for devices which are variously installed in the fire hose or at the fire hydrant. One of these devices is a valve controlled either by a conventional timer or radio control unit to open the valve subsequent to the attachment of the valve with hose to the hydrant. The other of these inventions pertains to a unique erodable element disposed in a special housing in the hose coupling and which is destroyed by the water after a period of time to permit full flow, the remains of the destroyed disc being being blown out the end of the hose. A pressure regulator valve operating on a principle similar to applicant's valve is shown in U.S. Pat. No. 1,097,879. This valve would not work in the present application, not only because of its delicacy, but also because it lacks the two distinct modes of operation necessary in the instant valve. The radio or timer controlled model described above represents the most sophistocated of the line but is not only quite expensive, but is rather complicated and thus subject to a certain amount of unavoidable human error and malfunction. This unit would also be inappropriate for small fire companies operating on a limited budget. The errodable disc was developed for these small companies, and as the disc requires replacing after every use but the adaptor coupling is simple, it is ideal for small fire companies which do not have many fires because of the minimal expense involved. However applicant felt there is a need for an intermediate device which although being more sophistocated than the errodable disc, is simpler, less expensive and most of all less subject to occasional human error than is the more complicated radio controlled model. SUMMARY OF THE INVENTION The delay valve of the present invention fulfills the above-mentioned need and operates automatically, once set into action, by the pressure drop across the valve chamber. In other words, when the hose is initially connected to the hydrant via the instant valve, the hydrant pressure is great compared to the absent pressure in the evacuated hose, and this pressure differential forces a hinge gate across the valve outlet. This gate is prevented from completely closing by means of a variable cam so that despite the pressure differential a slight flow is permitted so that the hose will, over a period of time, fill. Once the hose is filled, the pressure differential no longer exists and built-in spring bias snaps the gate open to permit free flow of water through the valve when the water is needed at the nozzle. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a side elevation view of the valve unit attached to a fire hydrant; FIG. 2 is an enlarged end view of the unit, as taken from the right hand side of FIG. 1; FIG. 3 is a sectional view taken on line 3--3 of FIG. 2; FIG. 4 is a sectional view taken on line 4--4 of FIG. 3, with the valve element in the near closed position; FIG. 5 is a sectional view taken on line 5--5 of FIG. 3; FIG. 6 is a side elevation view, partially cut away to show the valve element in full open position; FIG. 7 is a sectional view similar to FIG. 4, but showing an alternative dual inlet configuration; FIG. 8 is a side elevation view of the structure of FIG. 7; and FIG. 9 is a sectional view taken on line 9--9 of FIG. 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT A portion of a fire hydrant is shown at 10. The valve of the present invention has an outer casing 12 comprised of two mating plenums or half-chambers 14 and 16. The casing is made in two halves for convenience only and these two halves are permanently joined together at flanges 18 by bolts or rivets 20. The first half 14 of the casing is coupled to the nipple of the fire hydrant by means of a conventional gland 22 and the other half of the casing carries a typical threaded male coupling 24 identical to that of the fire hydrant for the connection of the fire hose 26. In obvious fashion water would enter the internal chamber 28 defined by the casing through the inlet 30 and exit the chamber through the outlet 32 defined by the interior lip of the nipple 24 as is best seen in FIGS. 3 and 6. To interfere with the flow of water through the chamber 28 a gate 34 is provided which is mounted on a shaft 36 which is in turn journalled in the casing wall. One end of this shaft terminates in a bearing cup 38 and the other end extends through the side wall of the casing and a reinforcing bushing 40, the end being orthogonally bent to define a reset lever 42 as can best be seen from FIG. 2. Viewing FIG. 3, the gate 34 would fall substantially flush against the inner rim 44 which defines the valve outlet 32 but is prevented from doing so by the following structure. A length of leaf spring 46 is carried by a pair of support arms 48 in spaced relation from the free end of the gate as is best seen in FIGS. 3 and 5. This spring conflicts with a spacer element, preventing the gate from completely closing, the spacer element in the preferred embodiment taking the form of eccentric cam 50. This cam mounts on the end of a short shaft 52 projecting through and externally of the casing wall, sealed by an O-ring 54 and terminating in a lever-bearing control knob 56. This knob can be used to rotatively adjust the angular disposition of the cam, and thus vary the spacing of the gate from the outlet 32 when the gate is in its most closed position. The outer casing may have markings on it which register with the lever of the control knob 56 to index the variance of the gate limitation. The cam could be modified and be made radio controlled so that the slow flow of water could be initiated from the fire site. By utilizing the cam itself as a valve, whether radio controlled or manually operated, operations are possible. For example, by throwing the cam wide open, a segment of hose can be filled rather quickly by a fireman at the fire hydrant. In an alternative construction of the resilient member, a bumper of neoprene or other resilient material could be seated in the gate 34 and covered with a metallic leaf spring to minimize wear of the member caused by striking the cam. This construction has proven itself durable and free from the vibration-inducing qualities of some of the other types of springs tested. Turning to FIGS. 2 and 5 and returning to the reset lever 42 of the gate shaft, it can be seen that a spring 58 biases this lever so that the attached gate would ordinarily displace away from the valve outlet 32 and alongside the casing walls completely clear of the flow of water through the chamber. However to prevent this from happening a second spring loaded element in the form of a detent clip 60 is mounted on the casing side wall to hold the lever against the action of the spring so that the gate is close to the valve outlet but not in its final closed position. Thus, due to the spring loading of the clip 60, when the gate is moved to its most closed position the detent will spring clear of the external lever 42 so that under the action of the spring 58 the gate will open absent a counterpressure caused by an adverse pressure differential. The operation of the valve should be obvious from the above description of its parts. It must be borne in mind that the other end of the hose which connects to the hydrant connects to the fire truck and is shut off during the operation of the valve. Other hoses lead from the fire truck to the fire site proper and are nozzle-controlled by on-site firemen determining the draw on the main hydrant hose. When a fire truck arrives at the hydrant near the fire site, the instant valve, which would be connected to the end of a hose, is attached to the fire hydrant as shown in FIG. 1 with the reset lever 42 being engaged by the detent 60. The fire hydrant is then turned on and the impact of the tremendous water pressure within the valve chamber 28 snaps the gate against the valve outlet to the extent permitted by the spring 46 as it strikes the cam 50. The hose 26, of course, is empty and flacid and provides no back pressure, so that the pressure differential is quite great and the force against the gate is commensurately high. As the gate is thus closed as far as it is permitted to travel, the detent 60 snaps back against the side wall of the casing in the position shown in FIG. 4. Now, because the gate is not entirely closed, water from the high pressure zone within the chamber flows into the outlet as is shown in FIG. 3 which over a period of several minutes will fill the hose completely. When this occurs (that is, complete filling of the hose) the high pressure on both sides of the hinged gate 34 destroy the pressure differential and the gate will swing open into the position indicated in FIG. 6 under the action of the lever spring 58. The length of hose between the hydrant and the fire truck has thus been filled, the obstructions cleared, and pressure is now available at the truck. To reuse the device, all that is necessary is of course to reset the lever 58 and engage it with the detent 60. Also the operation of the cam control knob 56 should be noted. By adjusting this knob, the time delay in the filling of the hose is affected in an obvious fashion. To accomodate different hose lengths, which are ordinarily provided in several standard lengths for fire-fighting, the actual hose length could be marked on the casing wall adjacent the control 56 to provide fairly standard timing of hose fill for different lengths. This knob could also obviously be used to make adjustments where for any reason the delay time would be advantageously greater or shorter than usual. The leaf spring 46 represents a resilient element which could be other than a leaf spring, for example, an elastomeric substance. This element could obviously be omitted and the gate made to bear directly on the cam. By using a resilient bumper member here the gate will restrict flow roughly in proportion to the pressure differential across it so that variations in hose fill time caused by differences in hydrant pressure will be evened out to a great extent. These pressure differences can be substantial in different locations, even in the same town. The effect of the cam and the partially closed clapper or gate of the present invention is clearly to provide a restricted, but not completely obstructed, passageway from the inlet to the outlet of the valve despite the substantial closure of the main gate 34. This could be accomplished by means of an alternative bleed passageway or duct rooted externally of the valve from the inlet to the outlet so that the gate would be permitted to close flush against the rim 44. This construction is intended to be within the scope of the invention. Such a duct or bleed passageway could also be provided with a stop cock or graduated valve to vary the timing of the initial hose fill, and pressure sensitive means could be incorporated to vary the aperature restriction based on pressure differential to duplicate the effect of the leaf spring bumper 46 in the illustrated embodiment. It is the essence of the invention that a valve restriction be provided which operates solely on pressure differential across the valve, and by means which are sensitive to the abatement of the pressure differential, the valve is thrown open to provide complete nozzle pressure at the operational end of the hose. A modified embodiment of the valve is shown in FIGS. 7 through 9. In this modification the essential operating mechanism of the valve as described above is still set forth in the half-chamber or half shell 16 defining the outlet half of the casing 12. In this instance through this half-chamber is coupled to a modified bi-valve chamber 62 having two gland nut coupled inlets 64 each of which is provided with a check valve 66 in the form of a pair of spring loaded flaps mounted inside the chamber. This double inlet chamber is part of an existing coupling which of course does not use the flow retarding valve structure in the outlet as does applicant's. The dual inlet is used in the following circumstance. The firemen attach one of the inlets to a fire hydrant and begin drawing water from that hydrant. Because the second of the inlet valves will be checked closed the valve in this mode will operate as though there were only a single inlet and a single outlet. If, however, it is subsequently found that the pressure in the hydrant is not high enough to adequately supply the number of hoses that are being fed from the secondary hoses connected to the fire truck, a supplemental water supply can be hosed in from another hydrant and introduced into the half chamber 62 through the second inlet. In this fashion the water supply to the valve outlet is continuous and uninterrupted while the additional pressure is coupled in. That same functioning of the double inlet coupling in retained in the structure shown in FIGS. 7 through 9 with the addition of applicant's flow retarding mechanism in the outlet half shell. By connecting the half shell 62 to the flow retardant half shell 16 by means of the flanges 18, which could be bolted together as well as riveted, the chamber 16 can be used alternatively with the double inlet half casing 62 and the single inlet half 14 already described. Although ordinarily there would not be a lot of shuffling in a fire house to disconnect the two halves and couple them to accomplish a different function, a great deal of retooling at the point of manufacture can be saved by utilizing the same coupling structure in both of the embodiments. Another feature of the invention which is shown only in the embodiment represented in FIGS. 7 through 9 for the sake of simplicity, but could equally well, and probably will be, used in the other version of the valve is the handle 68 best seen in FIG. 9. This handle projects from the side of the casing on which the reset lever 42 and its associated spring mechanisms are attached to protect this exterior structure from damage in handling. In its simplest form this handle is provided by extending the mating flanges of the half chambers 16 and 52 to include an opening 70 which is engageable easily by the hand. A small detaining leaf spring or the equivalent 72 may be mounted in the handle to detain the reset lever in its open mode as shown in phantom in FIG. 8. The purpose of this spring is to prevent the lever from accidentally being moved while the valve is in use, thus causing the gate to close and shutting off a supply of water to the firefighters. This spring as well as the handle, as mentioned above, could and probably would be incorporated in both embodiments of the invention. | Summary: The invention is a flow delay valve to be used in conjunction with fire fighting and attaches to the fire hydrant so that as the fire truck proceeds to unroll hose on the way to the fire site the hose is filled slowly due to the action of the delay valve, permitting the fire truck to proceed unhindered by full hose pressure but without requiring the stationing of a fireman at the hydrant to turn the hydrant on after the fire truck has arrived at the site as the valve fully opens automatically upon the hose becoming filled to permit unobstructed water flow. | 3,550 | 125 | big_patent | en |
Summarize: FIELD OF THE INVENTION The present invention relates to obtaining bioelectric information. In particular, the present invention relates to systems and methods for providing and using an enhanced tip to facilitate locating and obtaining a bioelectric resistance value from a patient for assessment, therapeutic and/or diagnostic purposes. BACKGROUND AND RELATED ART Traditional medical science has long recognized certain electrical characteristics of humans and other living organisms. For example, the traditional medical community has recognized the electrical potentials generated by the human body in such forms as brain waves as detected by electro-encephalographs (EEG), electrical impulses resulting from muscular heart activity as detected by electrocardiograms (EKG), and other electrical potentials measurable at other areas of the human body. While the relative levels of the electrical activity exhibit relatively small levels, such signals are nonetheless measurable and consistent. In addition to measurable voltage levels, the human body and other mammalian organisms exhibit specific locations wherein the resistance value and the conductance value are relatively predictable for healthy individuals. The locations of anatomical dermal conductance points exhibit unique resistance values. Interestingly, the locations on the body exhibit a resistive reading of approximately 100,000 ohms and coincide with the body locations that correspond to the acupuncture points defined anciently by the Chinese. Indeed, Chinese medical practitioners were aware of the art of treating unfavorable health conditions through the use of needles that are used to pierce peripheral nerves to relieve pain. Electrical stimulation of these points provides similar results. Many acupuncture points are situated above major nerve trunks and have nerves within 0.5 centimeters of their location. Studies have indicated that many acupuncture points correspond to nerve innervations and trigger points. The acupuncture points are located under the skin (epidermis) and are accessed electrically through the skin either by the use of acupuncture needles or by using a probe tip pressed against the skin. As the outermost layer of epidermis (cornified layer) is less conductive, the probe tip mayor may not need a fluid or a type of electrode gel to enhance conductivity through the skin to the acupuncture point. Even so, the amount of pressure required to access the point can frequently invoke painful responses from the patient. The representative acupuncture points and their relationship with organs and life systems of the human body have been characterized into more than 800 points that are organized into approximately 14 basic meridians. The measurable state of these acupuncture points reflects the condition of the related meridians and therefore the health of organs and other functions of the human body. In the art of acupuncture, the acupuncture points are generally located at the extremity region of the hands and feet. As introduced above, the resistance value of healthy tissue at an acupuncture or conductance point is generally in the range of about 100,000 ohms. When such tissue is inflamed or infected, the conductivity is higher such that the measured resistance value appears lower than 100,000 ohms. Additionally, if the tissue is in a degenerative state, the conductivity is lower causing the resistance value to be higher. Systems have been implemented to measure the resistance value at acupuncture points and present resistive values to a clinician for use in diagnosing a condition. However, the traditional systems have proven difficult to use since the precise location of the points is difficult to pinpoint, often requiring a probe tip to be placed on a specific angle in relation with the surface of the patient. Further, the differences in the characteristics of each patient and each point of a given patient can cause a practitioner to obtain inaccurate and/or unrepeatable readings. Moreover, current technologies have caused pain and/or discomfort to patients. In some systems, a first device is used to locate the points and a second device is brought in contact with the point to perform the electro-dermal screening. While this technique is available, employing multiple devices introduces a potential for clinical error. Accordingly, other systems have been made available that include both a point finding function and an electro-dermal screening function. However, in every case the system used proves difficult to locate the points on the patient. And, the electro-dermal screening is compromised when the system does not accurately determine the points. Thus, while techniques currently exist that are used to locate a point on a patient, challenges still exist, such as inaccurate readings, unrepeatable readings, pain, discomfort, and the like. Accordingly, it would be an improvement in the art to augment or even replace current techniques with other techniques. SUMMARY OF THE INVENTION The present invention relates to obtaining bioelectric information. In particular, the present invention relates to systems and methods for providing and using an enhanced surface to facilitate locating and obtaining a bioelectric resistance value from a patient for assessment, therapeutic and/or diagnostic purposes. Implementation of the present invention takes place in association with an abrasive bristly conductive probe tip that is used to obtain a bioelectric value from a patient. The uniform array of abrasive bristles which have a conductive surface are able to simultaneously contact and/or puncture a surface layer of a patient's skin (e.g., the cornified layer of the epidermis) to enable and facilitate locating and obtaining a bioelectric resistance value from the patient. In one implementation, the tip is covered with a clean protective cover. The cover protects the probe tip from damage and foreign contaminates. In addition, the cover provides a grip for the user to easily handle the probe tip. The cover also gives a mechanical advantage for the user to connect the tip to a probe. These and other features and advantages of the present invention will be set forth or will become more fully apparent in the description that follows and in the appended claims. Those skilled in the art will appreciate that the methods and processes can be used in association with a variety of different bioelectric sensing devices, including patches, clips, and the like to provide an enhanced bioelectric sensing surface. Furthermore, the features and advantages of the invention may be learned by the practice of the invention or will be obvious from the description, as set forth hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS In order that the manner in which the above recited and other features and advantages of the present invention are obtained, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. Understanding that the drawings depict only typical embodiments of the present invention and are not, therefore, to be considered as limiting the scope of the invention, the present invention will be described and explained with additional specificity and detail through the use of the accompanying drawings in which: FIG. 1 is a side elevation view of the tip with the broken line showing of environmental association matter being for illustrative purposes only and forming no part of the claimed device; FIG. 2 illustrates another representative a side view of the probe tip showing the uniform array of the bristles and the plain surface; FIG. 3 illustrates an exploded side view of the probe tip and cover; FIG. 4 illustrates another a side view of the probe tip and cover; FIG. 5 illustrates an exploded cross section view of the probe tip and cover; FIG. 6 illustrates another configuration of the cover and the tip; FIG. 7 illustrates a cross section view of the probe tip and cover. Drawing -Reference Numbers 2 Probe tip 4 Probe 6 Isolation hood 8 Bristle 12 seal rim 14 Cover 18 Probe shaft 20 Distal end 22 Proximal end 24 Receiving cavity 26 Grip 28 Screw threads 30 Plain surface 32 Level surface DETAILED DESCRIPTION OF THE INVENTION The present invention relates to obtaining bioelectric information. In particular, the present invention relates to systems and methods for providing and using an enhanced surface that is used to facilitate locating and obtaining a bioelectric values from a patient for assessment, therapeutic and/or diagnostic purposes. In the flowing description of the invention, certain terminology is used for the purpose of reference only, and is not intend to be limiting. Terms such as “upper”, “lower”, “above”, and “below,” refer to directions in the drawings to which reference is made. Terms such as “inwards” and “outward” refer to directions towards and away from, respectively, the geometric center of the component described. Terms such as “side”, “top”, “bottom,” “horizontal,” “with in,” “inside,” and “vertical,” describe the orientation of portions of the component within a consistent but arbitrary frame of reference which is made clear by reference to the text and the associated drawings describing the component under discussion. Such terminology includes words specifically mentioned above, derivatives thereof, and words of similar import. Embodiments of the present invention take place in association with an abrasive bristly conductive probe tip that is used to obtain a bioelectric value. Some embodiments embrace a bioelectric probe using the probe tip that includes a device for measuring and recording the bioelectric value. The abrasive bristly conductive probe tip includes an array of uniform bristles such that a variety of the bristles are able to simultaneously contact and/or puncture the cornified layer of a patient's epidermis to enable at least one bristle to be in contact with the acupuncture point and to obtain a bioelectric resistance value from the patient. In the disclosure and in the claims the term “abrasive bristly probe tip” shall refer to an abrasive construction of a plurality of bristles, wherein at least one of the bristles is able to locate and/or obtain a bioelectric value of a patient. An example of an abrasive bristly conductive probe tip includes one or more materials and/or coatings such as: brass, brass alloy or pleated brass. In accordance with at least some embodiments of the present invention, a patient may undergo bioelectric therapy corresponding to a condition diagnosed at an anatomical dermal conductance point. The various anatomical dermal conductance points are typically located throughout a patient's hands and feet. The dermal conductance points or acupuncture points aid the clinician in assessing and/or diagnosing a patient's condition and pinpointing a particular disorder. In accordance with embodiments of the present invention, a patent's condition may be assessed and/or diagnosed using a device or equipment capable of measuring the resistance or likewise the conductance at anatomical dermal conductance points located throughout the hands and feet of the patient. Thus, while embodiments of the present invention embrace a variety of different systems having an abrasive bristly conductive surface, the following relates to a representative system that includes a bioelectric probe having a probe tip. The probe tip is placed on an anatomical dermal conductance point. The conductance value is measured between the probe and a ground bar, and is displayed on a conductance monitor or other output for evaluation by a clinician or practitioner. If the conductance value at a particular conductance point on the patient denotes an imbalance, the clinician may investigate the biological system meridian that corresponds to the conductance point presenting the imbalanced reading. Conversely, when a particular conductance point displays a balanced reading, the clinician thereafter measures the conductance at various other conductance points to properly assess and/or diagnose the condition of the patient. Upon evaluating the condition of the patient, such as an organ disorder or a biological system abnormality, the clinician selects a possible remedy for such a condition. Remedies may include providing a homeopathic remedy or a digital sequence that is known to exhibit a particular reaction on an individual. An electromagnetic energy source generates a frequency coded electromagnetic signal containing the digital sequence, which is broadcast or projected upon the patient. The frequency coded electromagnetic signal may take the form of several electromagnetic types, such as radio frequency (RF) signals, infrared (IR) signals or other electromagnetic projections. As illustrated in FIG. 1, a probe tip 2 connects to a probe 4. The probe tip 2 includes screw threads 28 located within the probe tip 2. The screw threads 28 are designed to mate with a complementary thread (not shown) on the probe 4, such that the probe tip 2 is securely fastened to the probe 4, but easily removed from the probe tip 2. Those skilled in the art will recognize that different methods such as friction, smaller thread or quick releasing mechanisms may attach the probe tip 2 to the probe 4. FIG. 2 illustrates a probe tip 2 that includes a distal end 20 and a proximal end 22. The distal end 20 is convex and may be wider than the proximal end 22. The convex nature of distal end 20 reduces the need for a precise location and/or a precise angle in relation with the dermal surface layer to locate and obtain the bioelectric resistance value. As described above, the proximal end 22 is configured for electronic coupling to a mechanism for reading the bioelectrical resistance value such as a probe 4 shown in FIG. 1. Located between the distal end 20 and the proximal end 22 is a probe shaft 18. The probe shaft 18 includes a seal rim 12 which is discussed below. The distal end 20 includes an array of bristles 8. The bristles 8 form a uniform array pattern across the face of the distal end 20. Those skilled in the art will appreciate that while the bristles 8 have been illustrated in a circular configuration, other embodiments embrace other non-circular configurations. Further, as provided herein, at least some of the embodiments of the present invention include a low density of bristles 8 having a few multiple points to enable an enhanced bioelectric sensing surface. Each bristle 8 is a hexagon shape. However, those skilled in the art will recognize that other geometric shapes may form the individual bristles 8. The height of each bristle 8 ranges from 0.001 millimeters to 1 millimeters. In some embodiments, the bristles 8 are random in length. In other embodiments, the bristles are more uniform. Further, in some embodiments one or more of the bristles puncture the cornified layer of the epidermis to obtain the bioelectric value(s). In other embodiments, the bristles 8 do not puncture the cornified layer and may optionally be used in combination with a material, such as water, to enhance obtaining the bioelectric value(s). The preferred material of the bristle 8 is brass, a brass alloy or brass plated metal. In practice, the entire probe tip 2 will be made of the same material as the bristles 8. The bristles 8 may be coupled, machined, etched, molded and/or otherwise manufactured to the probe tip 2 in a variety of manners. As illustrated in FIG. 7, the probe tip 2 is protected by a cover 14. The cover 14 includes a receiving cavity 24 and a grip 26. During storage of the probe tip 2, the probe tip 2 is placed within the receiving cavity 24. In addition, the cover 14 keeps foreign contaminates from contacting the bristles 8, thus allowing the bristles 8 to remain sterile. The cover 14 is made from any non-conductive material such as plastic or nylon. Accordingly, FIGS. 3-5 provide additional representative cover 14 configurations for use in association with the present invention. Those skilled in the art will appreciate that the cover 14 embrace a variety of other types of configurations. As shown in FIG. 2, the seal rim 12 is located near the mid-section of the probe shaft 18. The diameter of the seal rim 12 is great than the diameter of the probe shaft 18. Thus, the seal rim 12 extends out from the probe shaft 18. However, the diameter of the seal rim 12 is slightly larger than the diameter of the receiving cavity 24. When probe tip 2 is placed inside the receiving cavity 24, the seal rim 12 pushes against the sides of the receiving cavity 24, creating resistance between the probe tip 2 and cover 14 such that a deliberate force is required to remove probe tip 2. Located at the proximal end 22 of the probe tip 2 is at least one plain surface 30. A corresponding level surface 32 is located on the receiving cavity 24 of the cover 14. The plain surface 30 and level surface 32 are similar in size and shape. When probe tip 2 is placed inside the receiving cavity 24, the plain surface 30 and the level surface 32 connect. When the user screws the probe tip 2 onto a probe 4, the user applies a torque force to the cover 14. The cover 14 transfers the torque force to the probe tip 2 to allow the tip to screw onto the probe 4. The increased surface area created by the plain surface 30 and the level surface 30, allows the cover 14 to transfer a great torqueing force to the probe tip 2. In practice, at least three plain surfaces 30 and level surfaces 32 will be located on the probe tip 2 and receiving cavity 24. The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The described embodiments are to be considered in all respects only as illustrative and not restrictive. The scope of the invention is, therefore, indicated by the appended claims rather than by the foregoing description. All changes that come within the meaning and range of equivalency of the claims are to be embraced within their scope. | Summary: The present invention is a device for providing an enhanced bioelectric sensing probe tip to facilitate locating and obtaining a bioelectric resistance value from a patient for therapeutic and/or diagnostic purpose. One embodiment of the present invention is a probe tip that comprises a uniform array of bristles that contacts a surface layer of a patient's skin to enable the bioelectric probe tip to locate and obtain a bioelectric resistance value from the patient. Another embodiment of the invention is to provide a probe tip cover that prevents foreign material from contaminating the probe tip and to assist in attaching the probe tip to a probe. | 3,980 | 128 | big_patent | en |
Summarize: Ledell Lee maintains innocence in death of Debra Reese None Share This Story On February 9, 1993, 26-year-old Debra Reese was sexually assaulted and murdered in her own home. She was struck approximately 36 times with a tire thumper, a tool used by truck drivers to check their tire pressure. Her husband was a truck driver and had given her the tire thumper as a means of protection while he was on the road. An hour after the murder, Jacksonville police arrested Ledell Lee in connection with Reese’s murder after witnesses claimed they saw him walking down the street that Reese’s house was on. After his arrest and even after his conviction, Lee reiterated his innocence. Joseph Lucky was a young child when his mother was murdered 24 years ago. To Lucky, his mother was everything to him. He has spent most of life hoping for justice and, most of all, closure for the death of his mother. “When she was ripped from my life it started spiral I almost didn’t recover from,” Luck admitted, “and my family has lived in the shadow of this event our entire lives.” Joseph Lucky at Ledell Lee's clemency hearing At Lee’s final clemency hearing, Lucky called Lee the “embodiment of evil that should never have to exist in this world.” Lee has been called a super predator by prosecutors. After being charged with the murder of Reese, he became a suspect in other crimes. Lee was convicted for the rape of two Jacksonville women and was accused of the murder and rape of 22-year-old Christine Lewis. In November 1989, Lewis was abducted from her home where she was later raped, strangled, and eventually killed. Her body was later found at an abandoned home inside a closet. That trial ended in a hung jury. When Lee received the death sentence for Reese’s murder, prosecutors decided not to pursue retrying him for the alleged murder of Lewis. Lee’s lawyer, Lee Short, said Lee’s past trials would have looked differently if “he was to have attorneys that didn’t have a conflict.” According to an Arkansas Supreme Court hearing, his lawyer was “so impaired by alcohol” during the first post-conviction trial that he had to be drug tested. Lee’s lawyer even admitted to being drunk during the post-conviction proceeding and exhibited compromised, bizarre behavior. During the trial, the lawyer was at one point unable to locate the witness room and was not familiar with his own witnesses. At one point, the lawyer rambled incoherently, interjecting “blah, blah, blah” during his statements. His behavior became so worrisome that the State of Arkansas’s counsel even admitted that the lawyer was submitting the “same items of evidence over and over again.” That lawyer was subsequently thrown out and new counsel was provided to Lee. Though the state has painted Lee as a super predator throughout his convictions, Lee’s brother knows a very different man. “He’s a great brother,” said Kevin Young. “He took me everywhere, taught me how to play basketball.” While Young now lives out of state, he said he has kept in close contact with his brother and has stood by him and believes Lee is innocent. Young claimed that Lee has not been given a fair trial. “We’re a praying family, so we’ve been hoping over the years that something would break and people would see that he’s innocent,” Young said. Brother of Ledell Lee believes he did not murder Debra Reese As Lee’s execution date drew near, the American Civil Liberties Union and the Innocence Project filed new motions which they hoped would help him prove he did not commit the murder of Reese 24 years ago. The ACLU asked for new post-conviction DNA testing in the case, and claimed that Lee’s intellectual disability stemming from fetal alcohol syndrome was “never noticed let alone brought to the court or the jury’s attention.” They asked that the court allow new DNA testing of a hair found at the scene and a drop of blood found on Lee’s shoe. Originally, the state’s experts said at the trial that the hairs matched Lee’s through microscopic examination. But both the ACLU and Innocence Project say that “forensic method” has now been discredited as non-factual and un-scientific. “The testing available then wasn’t sophisticated enough for fragmented samples,” ACLU said in a statement, “and no other DNA evidence was found to tie him to the crime.” However, Pulaski County Circuit Court Judge Herbert Wright denied the motion, saying that even if the court allowed the testing and it proved neither the hair nor the blood tied Lee to the crime, “there would still be sufficient proof presented by the state at trial for the jury to reach a guilty verdict.” That denial led to the Innocence Project joining the ACLU on the case. Both groups appealed the new DNA testing to the Arkansas Supreme Court and asked the court for a stay of Lee’s execution. “The state is rushing to put him to death without giving him the opportunity to do the DNA testing that could prove who actually committed the crime,” said Nina Morrison, a senior staff attorney with the Innocence Project. Despite these court battles, the family of Debra Reese hoped that his execution will go through as scheduled. And Lee’s family continued to fight until the last minute to prove his innocence. As for the State of Arkansas, they stayed the course, preparing to give Reese’s family the closure and peace of mind the law has promised. Lee was executed on Thursday, April 20 at 11:56 p.m. (CNN) Arkansas executed convicted murderer Ledell Lee, the first inmate to be put to death in the state in more than a decade. Lee, 51, was administered the lethal injection at 11:44 p.m. Thursday (12:44 a.m. ET). He was pronounced dead 12 minutes later. His execution was Arkansas' first since 2005. The state had planned to execute eight inmates over 11 days starting April 17, saying its lethal-injection drugs will expire at the end of the month. Four of the men have received stays for various reasons. Lethal injection Attorneys for the eight men attempted to block the executions, including using the argument that midazolam, the drug used to make inmates unconscious before two more drugs paralyze and kill them, does not effectively prevent a painful death. The second drug, pancuronium bromide, paralyzes the inmate. The third, potassium chloride, brings on cardiac arrest and stops the heart. "Unless the prisoner is unconscious, then drugs two and three will cause pain -- torturous punishment, in violation of the Eighth Amendment, and state guarantees against cruel and unusual punishment," said Jeffrey Rosenzweig, an attorney for three of the inmates. The lawyers have also fought the state's decision to conduct the series of executions in a short time frame, which the state has said is because its lethal-injection drugs will expire at the end of the month. "No state has ever conducted eight executions" over an 11-day period, according to Robert Dunham, executive director of the Death Penalty Information Center. One of the drug companies was granted a restraining order after arguing in court that their drug wasn't supposed to be used for capital punishment. McKesson Medical Surgical Inc. argued its vecuronium bromide was intended only for medical purposes, not executions, and that the Arkansas Department of Corrections "misled" the company when it purchased the drug, according to a court brief. "ADC (the Arkansas Department of Correction) personnel used an existing medical license, which is to be used only to order products with legitimate medical uses, and an irregular ordering process to obtain the vecuronium via phone order with a McKesson salesperson," the brief said. Two other inmates -- Jack Harold Jones and Marcel W. Williams, both sentenced to death for capital murder -- are scheduled to be executed Monday. Thirty-one states currently administer the death penalty, and lethal injection is the primary means of execution in all of them. The number of executions carried out in the United States fell to a 25-year low in 2016. Lee's conviction Lee was convicted in 1995 in the murder of Debra Reese, 26, two years prior. Reese was found dead in her home in Jacksonville, Arkansas, where she had been strangled and beaten with a small wooden bat her husband had given her for protection. Several of Reese's neighbors saw Lee near the house and identified him to police. Before Lee's death, all executions were put on hold in the state after a judge issued a restraining order on a key lethal injection drug. His execution followed a flurry of court rulings Thursday, capped by the US Supreme Court's denial of multiple requests for stays of execution. "Today is a shameful day for Arkansas, which is callously rushing the judicial process by treating human beings as though they have a sell-by date," Amnesty International said in a statement. "While other states have increasingly come to the conclusion that the capital punishment system is beyond repair, Arkansas is running in the opposite direction from progress. This assembly line of executions must stop and this cruel and inhuman punishment should be ended once and for all." At one point on Thursday night, the United States Supreme Court briefly delayed the execution as it reviewed appeals. It then voted to allow the state to proceed with the execution. About 30 minutes after the high court's ruling, Lee was pronounced dead. No final words Just before Lee was put to death, officials at the Cummins Unit prison in Grady, Arkansas, asked him twice for his final words, but he did not respond. His last meal was Holy Communion. At 11:44 p.m., Lee was given the lethal injection. His eyes closed three minutes later. He did not appear to show signs of discomfort, according to Sean Murphy, a reporter with the Associated Press who attended the execution as one of three media witnesses. "A member of the execution team began a consciousness check at 11:49 p.m. by flicking (Lee's) head, rubbing his eyes and tapping his sternum. Then at 11:55 p.m. his heart was checked with a stethoscope," Murphy said. Lee was officially pronounced dead one minute later. This undated photo provided by the Arkansas Department of Correction shows death-row inmate Ledell Lee. A ruling from the state Supreme Court allowing officials to use a lethal injection drug that a supplier... (Associated Press) VARNER, Ark. (AP) — The Latest on Arkansas' efforts to carry out executions before the end of April (all times local): 12:05 a.m. An Arkansas prison spokesman says the lethal injection of Ledell Lee took 12 minutes in the state's first execution since 2005 and its first involving a drug used in flawed executions in other states. Arkansas Department of Correction spokesman Solomon Graves says Lee did not make a final statement before his execution began at 11:44 p.m. Thursday. Graves says Lee was pronounced dead at 11:56 p.m., four minutes before his death warrant was due to expire. It was the first time Arkansas has used the sedative midazolam, and the state plans at least three more executions next week before its supply of the drug expires. ___ 11:56 p.m. Arkansas has executed an inmate for the first time in nearly a dozen years as part of its plan to execute several inmates before a drug expires April 30, despite court rulings that have already spared three men. Ledell Lee was pronounced dead at 11:56 p.m. Thursday. The 51-year-old Lee was given the death penalty for the 1993 death of his neighbor Debra Reese, whom Lee struck 36 times with a tire tool. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first three executions were canceled because of court decisions. Two more inmates are set to die Monday, and one on April 27. Another inmate scheduled for execution next week has received a stay. ___ 11:50 p.m. Dozens of death penalty opponents are gathered outside Arkansas Gov. Asa Hutchinson's mansion as the state prepares to conduct its first execution since 2005. The execution of Ledell Lee will be the first for Hutchinson as governor, who was elected in 2014. State prison officials moved forward with the execution after the U.S. Supreme Court lifted a temporary stay late Thursday. The protesters stood outside the gates of Hutchinson's home in Little Rock, holding candles and anti-death-penalty signs. ___ 11:30 p.m. The U.S. Supreme Court has cleared the way for Arkansas to conduct its first execution in nearly a dozen years. Justices rejected requests to stop Thursday night's execution of Ledell Lee. Prison officials were moving forward with plans to execute Lee before his death warrant expires at midnight. Lee was sent to death row for the 1993 beating death of his neighbor, Debra Reese. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 9:20 p.m. A federal appeals court has denied all requests for a stay from an Arkansas inmate set for execution Thursday night. That means Ledell Lee's fate now rests with the U.S. Supreme Court, which earlier in the evening rejected two requests for stays in separate cases. If executed, Lee would be the first Arkansas inmate put to death in nearly a dozen years. Lee's attorneys have filed challenges on a number of fronts, but so far, courts have ruled against the inmate. A temporary stay remains in place while the U.S. Supreme Court considers his appeals. Lee's death warrant expires at midnight. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 8:45 p.m. A federal appeals panel has rejected an inmate's request to stop his execution, set for Thursday night, as Arkansas aims to put an inmate to death for the first time since 2005. A three-judge panel of the 8th U.S. Circuit Court of Appeals denied Ledell Lee's request for a stay. Lee's attorneys had argued that he has a likely intellectual disability that has never been properly evaluated. Lee was initially set for execution at 7 p.m. Thursday, but the appeals court delayed that until 9:15 p.m. as it considered his case. Earlier, the U.S. Supreme Court rejected appeals in two unrelated cases that would have stopped Lee's execution, but he has other appeals pending with the high court. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 8:15 p.m. Courts have delayed for another hour the execution of an Arkansas inmate set to die Thursday night. Justice Samuel Alito issued a temporary stay until 8:30 p.m. CDT as the Supreme Court considers Ledell Lee's requests for a stay, while the 8th U.S. Circuit Court of Appeals issued a separate temporary stay until 9:15 p.m. Thursday as it considers a separate appeal. Lee's attorneys have filed a flurry of appeals in hopes of stopping his execution, which would be Arkansas' first in nearly a dozen years. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 8:05 p.m. An inmate facing execution in Arkansas on Thursday night has filed more emergency appeals with the U.S. Supreme Court to stop his lethal injection. Attorneys for Ledell Lee filed the requests late Thursday. Earlier, the U.S. Supreme Court rejected his appeals in separate cases. A federal appeals court has issued a temporary stay until 8:15 p.m. as it considers another of Lee's appeals. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 7:30 p.m. A divided Supreme Court has rejected appeals that would have put off the execution of Arkansas inmate Ledell Lee. More appeals to the high court were possible Thursday night. The four liberal justices dissented from a court order late Thursday that would not only have stopped Lee's execution, but also three more the state intends to carry out before the end of the month. That's when its supply of a key execution drug expires. New justice Neil Gorsuch voted with the majority of five to deny the stay of execution sought by Lee and the other inmates. Justice Stephen Breyer said he was troubled that the main reason for wanting to proceed with the executions was the looming expiration of the drug midazolam. Breyer asked, "Why these eight? Why now?" Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 7 p.m. A federal appeals court has delayed an Arkansas inmate's execution for at least an hour to review his request to stop his lethal injection scheduled for Thursday night. The 8th U.S. Circuit Court of Appeals temporarily stayed Ledell Lee's execution until 8:15 p.m. Thursday as it reviews his appeal. Lee had been set for execution at 7 p.m. Thursday. Lee's execution would be Arkansas' first since 2005. ___ 6:05 p.m. A federal appeals court has rejected a request to issue execution stays for a group of Arkansas inmates, including one scheduled for lethal injection Thursday night. The 8th U.S. Circuit Court of Appeals on Thursday rejected requests from the inmates, who had challenged the clemency process they underwent after Gov. Asa Hutchinson scheduled eight executions to take place over an 11-day period. Those challenging the policy include Ledell Lee, who is scheduled for execution Thursday night. He has other legal challenges pending but there are no stays in place that would stop his execution, which would be Arkansas' first since 2005. Arkansas set the compressed timetable because one of its lethal injection drugs expires April 30. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 5:45 p.m. A state prison official says an Arkansas inmate set for execution Thursday night declined a last meal and instead received communion. Inmate Ledell Lee is scheduled for execution at the Cummins Unit in southeast Arkansas. There are no stays blocking his execution, but he has several legal challenges pending. Arkansas Department of Correction spokesman Solomon Graves says Lee received communion Thursday afternoon instead of having a last meal. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of a lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 5:40 p.m. The Arkansas Supreme Court has denied another stay request from a condemned killer facing execution Thursday night. Justices on Thursday denied the request to halt the planned execution of Ledell Lee. Lee and Stacey Johnson had been scheduled to die Thursday night, but the state canceled plans to execute Johnson in the face of legal challenges. Lee has at least one stay request pending in a federal appeals court. The ruling was the third stay request for Lee denied by the Arkansas Supreme Court Thursday. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of another lethal injection drug expires. The first three executions were canceled because of court decisions. Legal rulings have put at least one other in doubt. ___ 5:20 p.m. Arkansas says one of two executions scheduled for Thursday night won't go ahead The Arkansas attorney general's office says it won't appeal an order halting the execution of Stacey Johnson. The decision by Attorney General Leslie Rutledge to not appeal Johnson's stay leaves only one inmate facing execution Thursday night. The state Supreme Court has denied two stay requests from Ledell Lee, but his attorneys are fighting on other fronts in state and federal court to halt his execution. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of another lethal injection drug expires. The first two executions were canceled because of court decisions. Legal rulings have put some of the others in doubt. ___ 4:50 p.m. The Arkansas Supreme Court says it won't reconsider its decision to halt the execution of an inmate the state planned to put to death Thursday night. In a 4-3 ruling, justices denied the state's motion to reconsider the stay issued for Stacey Johnson. His execution was halted after his attorneys sought additional DNA tests they say could exonerate him. It was unclear whether the state would appeal Johnson's stay to the U.S. Supreme Court. Arkansas had planned to execute Johnson and Ledell Lee Thursday night. Lee is fighting in federal and state courts for a similar stay. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of another lethal injection drug expires. The first two executions were canceled because of court decisions. Legal rulings have put the others in doubt. ___ 4:15 p.m. The Arkansas Supreme Court is allowing the state to use a lethal injection drug in upcoming executions, despite a supplier's complaint that it was sold to the state to be used only for inmates' medical care. Justices on Thursday lifted a judge's order preventing the state from using its supply of vecuronium bromide, one of three drugs used in Arkansas' lethal injection protocol. McKesson Corp., a medical supply company, said the state misleadingly bought the drug and that it wasn't intended for executions. The ruling clears one of the main legal hurdles the state faces in its effort to carry out two executions Thursday night. A stay remains in place for one of the inmates on an unrelated issue. Arkansas has not put an inmate to death since 2005. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of another lethal injection drug expires. The first two executions were canceled because of court decisions. Legal rulings have put the others in doubt. ___ 2:30 p.m. Two pharmaceutical companies that have said they don't want their drugs used in Arkansas' executions are asking to intervene in the court fight over whether the state can use a third lethal injection drug. Fresenius Kabi USA and West-Ward Pharmaceuticals Corp. asked Thursday to file a friend of the court brief urging the court to uphold an order preventing Arkansas from using its supply of vecuronium bromide, one of three drugs used in the lethal injection process. A supplier has said it sold the drug to Arkansas to be used for medical purposes, not executions. The companies have said they believe they manufactured the other two drugs Arkansas has for the executions, which are set for Thursday night. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of one lethal injection drug expires. The first two executions were canceled because of court decisions. Legal rulings have put the others in doubt. ___ 2 p.m. The Arkansas Supreme Court has rejected a stay request from an inmate as it seeks to hold its first executions since 2005, though another court decision has all executions scheduled in the state on hold. Justices on Thursday rejected a stay request from Ledell Lee. Their one-paragraph order did not elaborate on why. Lee is one of two inmates who had been set to die Thursday night. A judge in Little Rock has blocked the state from using one of the drugs in Arkansas' execution protocol because a company says the state misled it into providing the drug for lethal injections. Arkansas had scheduled eight executions over an 11-day period before the end of April, when its supply of a different lethal injection drug expires. The first two executions were canceled because of court decisions. ___ 12:45 p.m. Lawyers for the state of Arkansas have started their appeal of a decision that would prevent its executioners from using one of the three drugs in its lethal injection protocol. Circuit Judge Alice Gray has stopped the state's use of vecuronium bromide until she can determine the rightful owner. A drug supply company says Arkansas obtained the drug under false pretenses. The judge filed her order Thursday after the state complained to the Arkansas Supreme Court that she was taking too much time. Once her order was in, the state filed a notice that it would appeal. Arkansas originally scheduled eight executions over an 11-day period. The first two executions were canceled because of court decisions. Legal rulings have put the others in doubt. ___ 12:15 p.m. Arkansas inmates set for a series of executions before the end of the month have filed a new request for stays. In court papers filed Thursday, they say any new judges assigned to their cases in a state court at Little Rock should have time to become familiar with their pleadings. The state Supreme Court reassigned death penalty-related cases from a judge who went to an anti-death penalty rally after issuing an order last week barring the state from using one of its execution drugs. Lawyers for the state have complained that the inmates and their lawyers are trying to run out the clock, as one of Arkansas' execution drugs expires at the end of April. Arkansas originally scheduled eight executions over an 11-day period. The first two executions were canceled because of court decisions. Legal rulings have put the others in doubt. ___ 11:25 a.m. Lawyers for the state of Arkansas are trying to light a fire under a judge who has been slow to file written paperwork involving a death penalty case. Circuit Judge Alice Gray on Wednesday ordered the state to not use one of its execution drugs in executions set for Thursday night and next week. She wants to hold a hearing later on who really owns them — the state of Arkansas or a medical supply company that says it mistakenly provided them to the state prison system. Arkansas wants to appeal Gray's order, but needs a written order from her to do so. Its lawyers filed paperwork with the state Supreme Court on Thursday morning asking it to order her to submit a formal order. ___ 1 a.m. An aggressive effort by the state of Arkansas to carry out its first executions since 2005 stalled for the second time this week as courts blocked two lethal injections planned for Thursday, prompting Gov. Asa Hutchinson to express frustration at legal delaying tactics. While the latest court rulings could be overturned, Arkansas now faces an uphill battle to execute any inmates before the end of April, when one of its lethal injection drugs expires. The state originally set eight executions over an 11-day period in April, which would have been the most by a state in such a compressed period since the U.S. Supreme Court reinstated the death penalty in 1976. But Arkansas has faced a wave of legal challenges. Four of the eight have been granted stays of execution. VARNER, Ark. — The State of Arkansas, dismissing criticism that it intended to rush too many prisoners to their deaths too quickly, on Thursday night carried out its first execution in more than a decade. Using a lethal injection drug that has been the subject of sharp constitutional debate, the state plans to execute three more men by the end of the month, before its supply of the chemical expires. Ledell Lee, who was condemned to death for the murder of Debra Reese more than 20 years ago in a Little Rock suburb, died at 11:56 p.m. Central time at the Cummins Unit, a prison in southeast Arkansas, after the reprieves he had won in federal and state courts were overturned. He received injections of three drugs: midazolam, to render him unconscious; vecuronium bromide, to halt his breathing; and potassium chloride, to stop his heart. State officials administered the lethal injection at 11:44 p.m., after Mr. Lee, who requested holy communion as his last meal, wordlessly declined to make a final statement. Sean Murphy, a reporter for The Associated Press who witnessed the execution, said Mr. Lee was not visibly uncomfortable as he was put to death. The prisoner, Mr. Murphy said, was not responsive when the authorities performed consciousness checks. An evening of appeals kept Mr. Lee, 51, alive as his death warrant neared its midnight expiration. The United States Supreme Court, as well as a federal appeals court in St. Louis, issued temporary stays of execution while they considered his legal arguments. In Little Rock, the Arkansas capital, Gov. Asa Hutchinson monitored developments at the State Capitol. | Summary: After 12 years and hundreds of hours of legal battles, the state of Arkansas has successfully taken a man's life. Ledell Lee, a convicted murderer who'd been on death row for more than 20 years, was executed late Thursday after the US Supreme Court decided not to intervene, CNN reports. Prison officials say the 51-year-old inmate, who denied killing 26-year-old Debra Reese in 1993, requested Holy Communion as his last meal and declined to make a final statement. Neil Gorsuch voted with the majority in the Supreme Court's 5-4 decision to allow the execution to proceed, reports the New York Times. Lee, whose death warrant expired at midnight, took 12 minutes to die and was pronounced dead at 11:56pm. Arkansas had planned to execute eight inmates before the end of April, but court decisions spared the first three men and put the execution of a fourth in doubt, the AP reports. The ACLU and the Innocence Project had called for post-conviction DNA testing in the case of Lee, who was also convicted of two rapes and suspected of a second murder. In the attack Lee was executed for, Reese was sexually assaulted before being hit 36 times with a tire-thumping tool that her husband, a truck driver, had given her to protect herself with when he was on the road. Her son, Joseph Lucky, was 6 years old when she was killed. At Lee's final clemency hearing, Lucky called him "the embodiment of evil," THV11 reports. | 6,833 | 362 | multi_news | en |
Summarize: A former Delaware middle school teacher has avoided jail after exchanging explicit photos and having sex with one of her 13-year-old students. Stephanie Seabury, then 22, was captured on surveillance video climbing into her car outside Fred Fifer III Middle School in Camden with the unidentified boy on February 26. She then drove the teenager to her home at Dover's Woodmill Apartments, where she had sexual intercourse with him. Several weeks later, on March 12, the boy complained to police. Scroll down for video. Sentenced to probation: Stephanie Seabury (pictured, left, in her mugshot and, right, at Fred Fifer III Middle School in Delaware) has avoided jail after exchanging explicit photos and having sex with one of her students. Scene: Seabury, then 22, was seen climbing into her car with the 13-year-old boy on February 26. She then drove him to her home at Dover's Woodmill Apartments (above), where she had sexual intercourse with him. During a subsequent investigation, officers uncovered a series of 'explicit phone message and photographs [sent] during the inappropriate relationship,' police told NBC. Seabury, who had taught English at the school since August last year, turned herself into police on April 28. She was charged with sexual abuse of a child by a person in a position of trust or authority. Last Wednesday, she pleaded guilty to one count of fourth-degree rape, Delaware Online reported. But on Monday, the 23-year-old escaped jail after being sentenced to 18 months' probation by Superior Court Judge, Robert B. Young. She must also register as a Tier II sex offender. Following her sentencing, reporters attempted to contact Seabury at her family home. A man who answered the door told them to call her lawyer, before adding: 'She's innocent.' Guilty: Seabury, who had taught English at the school since August last year, turned herself into police on April 28. Above, the Long Island, New York, native is pictured leaving the police station after handing herself in. According to police, investigators contacted officials at Fred Fifer III on March 13 - a day after the boy complained of being assaulted at Seabury's apartment in the 1300 block of S. Farmview Drive. Staff confirmed that the victim was one of Seabury's students. While working with police, they also located the surveillance footage showing the pair leaving the school together on the day of the rape. 'It was a very highly inappropriate relationship,' said Dover Police Corporal, Mark Hoffman, last week. 'Especially with a 13-year-old and a 22-year-old, and, of course, being a teacher.' Seabury, a Long Island, New York, native and University of Delaware graduate, has also been ordered to have no contact with either the victim or other children. School: During a police investigation, officers uncovered a series of 'explicit phone message and photographs [sent] during the inappropriate relationship' between Seabury and the student at Fred Fifer III (above) | Summary: Stephanie Seabury, then 22, caught on video climbing into car with boy. She then drove the 13-year-old to her home, where she had sex with him. During investigation, police discovered 'explicit messages and photos' English teacher admitted to one count of fourth-degree rape last week. Handed 18 months' probation and made to sign sex offenders' register. Victim was one of her students at Fred Fifer III Middle School, Delaware. | 725 | 107 | cnn_dailymail | en |
Summarize: Introduction This report provides a broad overview of U.S. immigration policy. The first section addresses policies governing how foreign nationals enter the United States either to reside permanently or to stay temporarily. Related topics within this section include visa issuance and security, forms of quasi-legal status, and naturalization. The second section discusses enforcement policies both for excluding foreign nationals from admission into the United States, as well as for detaining and removing those who enter the country unlawfully or who enter lawfully but subsequently commit crimes that make them deportable. The section also covers worksite enforcement and immigration fraud. The third section addresses policies for unauthorized aliens residing in the United States. While intended to be comprehensive, this primer may omit some immigration-related topics. It does not discuss policy issues or congressional concerns about specific immigration-related policies and programs. Immigration Inflows and Related Topics U.S. immigration policy is governed largely by the Immigration and Nationality Act (INA), which was first codified in 1952 and has been amended significantly several times since. Implementation of INA policies is carried out by multiple executive branch agencies. The Department of Homeland Security (DHS) has primary responsibility for immigration functions through several agencies: U.S. Citizenship and Immigration Services (USCIS), Customs and Border Protection (CBP), and Immigration and Customs Enforcement (ICE). The Department of State (DOS) issues visas to foreign nationals overseas, and the Department of Justice (DOJ) operates immigration courts through its Executive Office of Immigration Review (EOIR). Foreign-born populations with different legal statuses are referred to throughout this report. The term a liens refers to people who are not U.S. citizens, including those legally and not legally present. The two basic types of legal aliens are (1) immigrants (not including refugees and asylees) and (2) nonimmigrants. Im mi grant s refers to foreign nationals lawfully admitted to the United States for permanent residence. N onimmigrant s refers to foreign nationals temporarily and lawfully admitted to the United States for a specific purpose and period of time, including tourists, diplomats, students, temporary workers, and exchange visitors, among others. Refugees and asylees refer to persons fleeing their countries because of persecution, or a well-founded fear of persecution, on account of race, religion, nationality, membership in a particular social group, or political opinion (see " Refugees and Asylees "). Refugees and asylees are not classified as immigrants under the INA, but once admitted, they may adjust their status to lawful permanent resident (LPR). Na turalized citizens refers to LPRs who become U.S. citizens through a process known as naturalization (described below), generally after residing in the United States continuously for at least five years. Noncitizen s are persons who have not naturalized and may include immigrants as well as nonimmigrants. U nauthorized alie ns refers to foreign nationals who reside unlawfully in the United States and who either entered the United States illegally ("without inspection") or entered lawfully and temporarily ("with inspection") but subsequently violated the terms of their admission, typically by "overstaying" their visa duration. Permanent Immigration5 Four general principles underlie the current system of permanent immigration: family reunification, U.S. labor market contribution, origin-country diversity, and humanitarian assistance. These principles are reflected in different components of permanent immigration. Family reunification occurs primarily through family-sponsored immigration. U.S. labor market contribution occurs through employment-based immigration. Origin-country diversity is addressed through the Diversity Immigrant Visa. Humanitarian assistance occurs primarily through the U.S. refugee and asylee programs. These permanent immigration pathways are discussed further below. The INA limits worldwide permanent immigration to 675,000 persons annually: 480,000 family-sponsored immigrants, made up of family-sponsored immediate relatives of U.S. citizens ("immediate relatives"), and a set of ordered family-sponsored preference immigrants ("preference immigrants"); 140,000 e mployment-based immigrants ; and 55,000 diversity visa immigrants. This worldwide limit, however, is referred to as a "permeable cap" because immediate relatives are exempt from numerical limits placed on family-sponsored immigration (described below) and thereby represent the flexible component of the 675,000 worldwide limit. In addition, the annual number of refugees is determined not by statute but by the President, in consultation with Congress. Consequently, actual total annual LPRs (immigrants, refugees, and asylees) have averaged roughly 1 million persons during the past decade. To ensure that a few countries do not dominate permanent immigration flows, the INA further specifies a "per-country limit" or "cap" limiting the number of family-sponsored preference immigrants and all employment-based immigrants from any single country to 7% of the limit in each preference category. Family-Sponsored Immigration Family-sponsored immigration consists of two immigrant groups ( Table 1 ). I m mediate relative s include spouses and minor unmarried children of U.S. citizens and parents of adult U.S. citizens. An unlimited number of immediate relatives can acquire LPR status each year if they meet the standard eligibility criteria required of all immigrants. Pre ference immigrants, on the other hand, are numerically limited to 226,000 per year and, unlike immediate relatives, are also bounded by the 7% per-country limit. In recent years, family-sponsored immigrants have accounted for two-thirds of all permanent immigration. Employment-Based Immigration Employment-based immigration occurs through five numerically limited preference categories ( Table 2 ), the first three of which are ranked by professional accomplishment and ability. The fourth preference category includes various "special immigrants," and the fifth preference category includes immigrant investors (i.e., EB-5 visa holders), a category created in 1990 to benefit the U.S. economy through employment creation and capital investment. Employment-based immigrants include accompanying spouses and children of qualifying LPRs, are limited to 140,000 total annual admissions, and are subject to the same 7% per-country limit as family-sponsored preference immigrants. Visa Queue The number of foreign nationals seeking to immigrate to the United States each year through family-sponsored and employment-based preference categories typically exceeds the INA-mandated numerical limits. Prospective immigrants are further constrained by the 7% per-country cap that primarily impacts foreign nationals from countries that send many immigrants to the United States (e.g., Mexico, China, India, Philippines). As a result, many foreign nationals who meet the U.S. immigrant qualifications and whose petitions have been approved by USCIS must wait years before a numerically limited visa from DOS becomes available. When a visa becomes available, it allows an approved prospective immigrant to travel to the United States and, if admitted to the country by an immigration officer at a port of entry, receive LPR status. If the approved prospective immigrant already resides in the United States on a nonimmigrant visa, the availability of a visa allows him or her to "adjust status" (i.e., change from a temporary nonimmigrant to a permanent immigrant with LPR status) without having to return to the country of origin to complete visa processing through a DOS consulate. As of November 1, 2017, the queue of approved family-sponsored and employment-based immigrants waiting for visas numbered 4.1 million persons. Diversity Immigrant Visa The diversity immigrant visa fosters legal immigration from countries that send relatively few immigrants to the United States. Each year, 50,000 visas are made available to selected natives of countries from which immigrant admissions totaled less than 50,000 over the preceding five years. Since the visa's inception in the early 1990s, the regional distribution of diversity lottery immigrants has shifted from Western European to African and Eastern European countries. To be eligible for a diversity immigrant visa, foreign nationals must have a high school education or two years of work experience within the past five years in an occupation that requires at least two years of training or experience to perform. Diversity immigrant visa applicants are selected by lottery. Winners of the diversity immigrant visa lottery must also meet the standard eligibility criteria required for most immigrants. Refugees and Asylees The United States has long held to the principle that it will not return a foreign national to a country where his or her life or freedom would be threatened. This is embodied in several INA provisions, notably in those defining refugees and asylees. A refugee is a person who is outside his or her home country (a second country that is not the United States) and is unable or unwilling to return because of persecution, or a well-founded fear of persecution, on account of five possible criteria: (1) race, (2) religion, (3) nationality, (4) membership in a particular social group, or (5) political opinion. An asylee is a person who meets the definition of a refugee in terms of persecution or a well-founded fear of persecution but who has been admitted in the United States or is present at a land border or port of entry to the United States. Refugee status is granted within numerical limits. Refugee admissions differ from other immigrant admissions because their annual number, known as the refugee ceiling, and their allocation by world region are not mandated in statute but set each year by the President, in consultation with Congress. For FY2018, the worldwide refugee ceiling was set at 45,000, allocated among world regions (Africa, East Asia, Europe and Central Asia, Latin America/Caribbean, and Near East/South Asia). Asylum is granted on a case-by-case basis and is not numerically limited. An alien in the United States may "affirmatively" apply to USCIS for asylum or may "defensively" seek asylum before an immigration judge during proceedings that determine an individual's removability under the INA. Typically, aliens arriving at a U.S. port of entry who lack proper immigration documents for admission or who engage in fraud or misrepresentation are placed in expedited removal (described below); however, if they express a fear of persecution, they receive a "credible fear" review by a USCIS asylum officer and—if found to have credible fear—are referred to an immigration judge for a hearing. Other Pathways to Lawful Permanent Resident Status In addition to the primary components of permanent immigration discussed above, there are several other pathways to LPR status, though they account for relatively few immigrants. The most prominent among these are c ancellation of r emoval, U nonimmigrant visas, and T status. Cancellation of r emoval is a discretionary, case-by-case form of relief granted by an immigration judge to aliens in removal proceedings. To receive it, an alien must demonstrate substantial ties to the United States, be of good moral character, and not have been convicted of a crime that makes him or her removable. More specific requirements differ by legal status. Because an immigration judge grants cancellation of removal at his or her discretion, no fixed standard exists for who merits relief. The alien must show that he or she is eligible for and deserves the relief. Grants of cancellation of removal are limited to 4,000 LPRs and 4,000 nonpermanent residents per year. U nonimmigrant visas are granted to certain victims who help law enforcement agencies investigate and prosecute domestic violence, sexual assault, human trafficking, and other crimes. After meeting specific requirements, U visa recipients, as well as their immediate family members, can acquire LPR status. The INA limits U visas to 10,000 per year. T status is granted to alien victims of severe forms of human trafficking. T status recipients may remain in the United States for four years and apply for LPR status after three. To qualify for T status, trafficking victims must also (1) be physically present in the United States, its territories, or a U.S. port of entry either because of such trafficking or to participate in related investigations or prosecutions; (2) have complied with requests to assist law enforcement investigating or prosecuting trafficking acts; and (3) be likely to suffer unusual and severe harm upon removal. Such aliens must also be admissible to the United States or obtain a waiver of inadmissibility. The INA limits T status to 5,000 principal aliens annually. Requirements for Permanent Admissions To obtain LPR status, prospective immigrants must traverse a multistep process through several federal departments and agencies. If they already reside legally in the United States, obtaining LPR status involves adjusting from a temporary nonimmigrant status to lawful permanent resident status. If they live abroad and have not established a lawful residence in the United States, their petitions are forwarded to DOS's Bureau of Consular Affairs in the home country after they have been adjudicated and approved by USCIS. The consular officer (when the alien lives abroad) and USCIS adjudicator (when the alien is adjusting status within the United States) must be satisfied the alien is entitled to the immigrant status. These reviews are intended to ensure that prospective immigrants are not ineligible for visas or admission under the INA's grounds for inadmissibility (see section on " Visa Issuance and Security "). Temporary Admissions Each year, the United States admits millions of nonimmigrants, including tourists, foreign students, diplomats, temporary workers, exchange visitors, internationally known entertainers, foreign media representatives, intracompany business personnel, and crew members on foreign vessels. DOS issues 87 specific types of nonimmigrant visas within 24 major nonimmigrant visa categories. Categories are often referred to by the letter and numeral denoting their subsection within INA Section 101(a) (e.g., B-2 tourists, E-2 treaty investors, F-1 foreign students). Requirements for Temporary Admission Foreign nationals who apply for temporary admission must demonstrate, both to DOS consular officers at the time they apply for a visa in their home countries, as well as to CBP officers when they apply for admission upon arrival in the United States, that they are eligible for both nonimmigrant status and the specific requested nonimmigrant visa. In addition, because the INA presumes that all aliens seeking admission to the United States are coming to live permanently, nonimmigrant applicants must demonstrate that they intend to stay for a temporary period and a specific purpose. With certain exceptions, nonimmigrants are prohibited from working in the United States. In recent years, the most numerous nonimmigrants entering the United States have included B-1 business visitors; B-2 tourists; Border Crossing Card holders; L intracompany transferees employed with international firms; H-1B professional specialty workers; H-2A agricultural guest workers and H-2B nonagricultural guest workers; J cultural exchange visitors (including professors, students, foreign medical graduates, teachers, resort workers, camp counselors, and au pairs); and F foreign students. Visa Waiver Program The Visa Waiver Program (VWP) allows nationals from certain (mostly high income) countries to enter the United States as temporary visitors for business or pleasure without obtaining visas from U.S. consulates abroad. Those entering the country under the VWP undergo a biographic (e.g., name, address, date of birth) rather than a biometric (e.g., fingerprint) security screening and do not need to be interviewed by a U.S. government official before their trip. In FY2016, roughly 18.7 million pleasure and 3.1 million business visitors entered the United States using the VWP. Border Crossing Card Mexican citizens who live in Mexico and who are admissible as B-1 business or B-2 tourist visitors can apply for a border crossing card (BCC) to gain short-term entry into the United States. In FY2017, roughly 1.1 million Mexican nationals were issued border crossing cards. The BCC may be used for multiple entries and is valid usually for 10 years. Current rules limit BCC holders to visits of up to 30 days within a zone of 25 miles along the border in Texas and California, 55 miles along the New Mexico border, and 75 miles along the Arizona border. U.S. admissions from persons possessing border crossing cards in FY2016 totaled 101.9 million. Visa Issuance and Security All nonresident foreign nationals who wish to come to the United States, whether permanently or temporarily, must obtain a visa. A visa permits a foreign national to travel to a U.S. port of entry and request permission from CBP to enter the country. To obtain a visa, foreign nationals must establish their qualification for a specific visa under its admission criteria. Visa issuances to the United States can be denied under three INA provisions: insufficient information under INA Section 221(g); the grounds of inadmissibility under INA Section 212(a); and for nonimmigrant applicants, the presumption of seeking permanent residence under INA Section 214(b). Visa applications must be complete. A visa denial under INA Section 221(g) indicates that the DOS consular officer abroad lacks sufficient information to determine if a foreign national is eligible to receive a visa. A consular officer may also disqualify a visa applicant if (1) he or she knows or has reason to believe that the alien is inadmissible under INA Section 212(a) (described below) or any other provision of law; or (2) the application fails to comply with INA provisions or regulations. All foreign nationals must undergo admissibility reviews. Consular officers must decide whether a foreign national is excludable under the grounds in INA Section 212(a), which include the following: health-related grounds; criminal grounds; security and terrorist concerns; public charge risk (e.g., indigence); seeking to work without proper labor certification; illegal U.S. entry and U.S. immigration law violations; ineligibility for U.S. citizenship; and having been previously removed from the United States. The INA describes procedures for making and reviewing an inadmissibility determination, and specifies conditions under which some of these provisions may be waived. For nonimmigrant applicants, a visa denial under INA Section 214(b) indicates that the foreign national was unable to demonstrate to the consular officer that he or she had sufficient ties to his or her home country to return home. This is the most common reason that DOS denies nonimmigrant visas. All visa applicants are required to submit their photographs, fingerprints, and biographic and demographic information. All prospective LPRs and certain prospective nonimmigrants must also submit to physical and mental examinations. Visa applicants are checked against multiple databases and information sources for security purposes. Consular officers' decisions on whether or not to grant foreign nationals a visa are not subject to judicial appeals. Types of "Quasi-legal" Status Two immigration mechanisms that allow persons to remain legally in the United States without being legally admitted into the country, Temporary Protected Status and parole, are described below. Other options, including Deferred Enforced Departure, withholding of removal, and deferred action, are described in later sections of this report. Temporary Protected Status When extraordinary conditions such as civil unrest, violence, or natural disasters occur in foreign countries, foreign nationals from those places who are present in the United States may not be able to return home safely. The INA allows DHS, in consultation with DOS, to grant Temporary Protected Status (TPS) to such foreign nationals, provided that doing so is consistent with U.S. national interests. Congress has also provided TPS legislatively. While TPS beneficiaries may obtain employment authorization, TPS does not provide a path to LPR status. DHS can designate TPS for 6 to 18 months and may extend it if conditions do not change in the designated country. Based on the most recent designations for each country, there are an estimated 317,600 TPS recipients from 10 countries: El Salvador, Haiti, Honduras, Nepal, Nicaragua, Somalia, Sudan, South Sudan, Syria, and Yemen. Parole DHS may, at its discretion and on a case-by-case basis, "parole" an alien into the United States for urgent humanitarian reasons or significant public benefit. Parole does not constitute formal admission to the United States and is not classified as a formal immigration status (e.g., nonimmigrant, immigrant). It is granted for a specified period of time. Parolees may obtain employment authorization but must leave when the parole expires or, if eligible, be admitted in a lawful status. Naturalization Under U.S. immigration law, all LPRs may become U.S. citizens through a process known as naturalization. With some exceptions, aliens must do the following to naturalize: reside continuously in the United States as LPRs for five years; demonstrate that they possess good moral character; demonstrate basic English skills and knowledge of U.S. history and civics; and take an oath of allegience to the United States. An estimated 43.7 million foreign-born persons resided in the United States in 2016, roughly divided between 21.2 million (49%) naturalized citizens and 22.5 million (51%) noncitizens. Immigration Enforcement Immigration enforcement encompasses enforcement of the INA's civil provisions (e.g., violations of admission conditions) as well as its criminal provisions (e.g., marriage fraud, alien smuggling). It involves border security where foreign nationals enter the United States (at ports of entry) and along U.S. borders (between ports of entry), as well as enforcing immigration laws in the U.S. interior, including worksite enforcement. Immigration enforcement also involves the identification, investigation, apprehension, prosecution, and deportation of foreign nationals who violate U.S. laws and become removable. Border Security at Ports of Entry Foreign nationals arrive in the United States at one of 329 ports of entry (POEs), including land checkpoints, airports, and seaports. They are met by CBP officers whose primary immigration enforcement mission is to ensure that such travelers are eligible to enter the United States and to exclude inadmissible foreign nationals. Possession of a visa by a foreign national does not guarantee U.S. admission if a CBP officer finds the individual inadmissible under law. About 390 million travelers (citizens and noncitizens) entered the United States in FY2016, including roughly 251 million land travelers, 119 million air passengers and crew, and 20 million sea passengers and crew. During the same period, about 274,000 aliens were denied admission and 21,000 aliens were arrested on criminal warrants. To balance facilitating the flow of lawful travelers with the competing interest of ensuring border security and immigration enforcement, DHS relies on a risk management strategy that includes screening at multiple points in the immigration process, beginning well before travelers arrive at U.S. POEs. DHS and other departments involved in the "inspection" process use screening tools to distinguish known, low-risk travelers who may be eligible for expedited admissions processing from lesser-known, higher-risk travelers who are usually subject to more extensive secondary inspections. DHS is also responsible for implementing an electronic entry-exit system at POEs, a task Congress mandated in 1996. While CBP collects a portion of the requisite biographic and biometric data from noncitizens at various stages of their entry to and exit from the United States, implementation of a fully automated biometric system has proven challenging. Border Security Between Ports of Entry Border security between POEs focuses on unauthorized land border entry into the United States, which has been a concern for Congress since the 1970s, when unauthorized migration first registered as a major national issue. It has received greater attention since the September 11, 2001, terrorist attacks. CBP's unauthorized migration control strategy between POEs has involved "prevention through deterrence," or concentrating personnel, infrastructure, and surveillance technology along heavily trafficked border regions. More recently, CBP's strategy has involved "enforcement with consequences," which includes making migrants who commit crimes face criminal charges and incur penalties (including incarceration) prior to removal. For Mexican nationals, this can include repatriation to Mexican locations geographically remote from where migrants were apprehended. Criminal charges, penalties, and formal removal generally bar migrants from legally entering the United States for varying lengths of time. Remote repatriation is intended to disrupt migrant smuggling networks and reduce the likelihood that removed migrants will reenter the country illegally. The United States has substantially increased appropriations for personnel, fencing, infrastructure, and surveillance technology for border enforcement over the last three decades, particularly after 2001. Since receiving authorization from Congress in 1996, DHS has built 653 miles of several types of barriers along the U.S.-Mexico border. CBP also employs land-based, aerial, and marine surveillance technologies. The most widely cited metric of border security is unauthorized migrant apprehensions, which are usually positively related to the flow of unauthorized migrants. Annual apprehensions were relatively low in the 1960s, climbed sharply after 1965, and reached peaks of roughly 1.7 million in both 1986 and 2000. They have fallen since then to 310,531 apprehensions in FY2017. The extent to which reduced inflows resulted from more effective enforcement rather than other factors, like the U.S. economic downturn in the late 2000s, remains subject to debate. Detention U.S. law provides broad authority to detain foreign nationals awaiting the outcomes of their removal proceedings. The law mandates detention for certain categories of aliens, including those arriving to the United States with fraudulent or no documentation; who are inadmissible or deportable on criminal or national security grounds; who are certified as terrorist suspects; or with final orders of deportation (with some limitations). Detention priorities are specified in statute and regulations and have been expanded legislatively in recent years. Other detained aliens include persons who are arrested for being illegally present in the United States. Most detained aliens are quickly returned to their country of origin through a process known as expedited removal (described below). Aliens not subject to mandatory detention may be either detained, paroled, or released on bond. DHS detained 352,880 noncitizens during FY2016. The amount of detention space, which has increased from 21,100 beds in FY2002 to 34,000 beds in FY2016, is controlled almost exclusively through congressional appropriations. In FY2016, almost all detained aliens had been prioritized for removal because they had committed specific felony crimes, multiple misdemeanors, or specific immigration violations targeted by ICE. The U.S. Supreme Court has ruled that the detention of unauthorized foreign nationals generally may not last beyond six months. In addition, ICE must obtain travel documents to repatriate foreign nationals. Because some countries refuse to provide such documents or do so in a protracted manner, ICE has regularly released sizeable numbers of detainees following their full detention term, including criminal aliens—an outcome that has been criticized repeatedly by some Members of Congress. Removal Removing foreign nationals who violate U.S. immigration laws is central to immigration enforcement, and the INA provides broad authority to DHS and DOJ to remove certain foreign nationals from the country. This includes unauthorized aliens as well as lawfully present foreign nationals who commit certain acts that make them removable. Any foreign national found to be inadmissible (either before or after U.S. entry) or deportable under grounds specified in the INA may be ordered removed. To remove a lawfully admitted alien, the U.S. government must prove that the noncitizen has violated one of the following six grounds of deportation specified in INA Section 237(a): being inadmissible at the time of entry or violating one's immigration status; committing certain criminal offenses, including crimes of "moral turpitude," aggravated felonies, alien smuggling, and high-speed flight from an immigration checkpoint; failing to register with DHS (if required) or committing document fraud; being a security risk (including violating any law relating to espionage, engaging in criminal activity that endangers public safety, partaking in terrorist activities, or assisting in Nazi persecution or genocide); becoming a public charge within five years of entry; or voting unlawfully. The INA describes procedures for making and reviewing a removal determination, and specifies conditions under which certain grounds of removal may be waived. DHS officials may exercise some discretion in pursuing removal orders, and certain removable aliens may be eligible for permanent or temporary relief from removal. Other grounds for removal (e.g., criminal, terrorist) render foreign nationals ineligible for most forms of relief and may make them subject to more streamlined (expedited) removal processes, both at the U.S. border and within the U.S. interior. Under the standard removal process, an immigration judge from EOIR determines in a civil judicial proceeding whether an alien is removable. The immigration judge may grant certain forms of relief (e.g., asylum, cancellation of removal), and removal decisions are subject to administrative and judicial review. Under streamlined removal procedures, including expedited removal and reinstatement of removal (i.e., when DHS reinstates a removal order for a previously removed alien), opportunities for relief and review are generally limited. Under expedited removal (INA §235(b)), an alien who lacks proper documentation or has committed fraud or willful misrepresentation to gain admission into the United States is inadmissible. That individual may be removed without any further hearings or review, unless he or she indicates an intention to apply for asylum. Two other removal options that are often referred to as "returns"— voluntary departure and withdrawal of petition for admission —require aliens to leave the United States promptly but exempt them from certain penalties associated with other types of removal. Following an order of removal, an alien is generally ineligible to return to the United States for a minimum of five years and possibly longer depending on the reason for and type of removal. Absent other factors, unlawful presence in the United States is a civil violation, not a criminal offense, and removal and its associated administrative processes are civil proceedings. As such, aliens in removal proceedings generally have no right to free government-provided counsel, although they may obtain counsel at their own expense. Programs Targeting Criminal Aliens Although all unauthorized aliens are potentially subject to removal, the removal of criminal aliens (noncitizens who have been convicted of a crime in the United States) has been a statutory priority since 1986. Programs targeting criminal aliens for removal have grown substantially since DHS was established in 2002. ICE currently operates several programs that identify and remove criminal and other removable aliens, including the Criminal Alien Program (CAP), an umbrella program for coordinating the agency's resources. CAP includes a data sharing infrastructure, or "interoperability," between DHS and DOJ that screens for both immigration and criminal violations when individuals are booked into jail. To pursue known at-large criminal aliens and fugitive aliens outside of controlled settings (i.e., administrative offices or custodial settings), ICE uses the National Fugitive Operations Program (NFOP). In addition to programs using DHS personnel, ICE's Section 287(g) program allows DHS to delegate certain immigration enforcement functions to specially trained state and local law enforcement officers, under federal supervision. Options for Aliens in Removal Proceedings Provisions in the INA permit certain removable aliens to remain in the United States, either permanently or temporarily. Options that provide permanent relief by conferring or leading to lawful permanent resident status include cancellation of removal (discussed above) and "defensive" asylum applied for during removal proceedings. Options that provide temporary relief include withholding of removal, the Convention Against Torture, Deferred Enforced Departure, and deferred action (described below). Worksite Enforcement The INA prohibits the employment of individuals who lack work authorization. Its provisions, sometimes referred to as employer sanctions, make it unlawful for an employer to knowingly hire, recruit or refer for a fee, or continue to employ an alien who is not authorized to be so employed. ICE's worksite enforcement program primarily targets cases involving critical infrastructure or employers who commit "egregious" violations of criminal statutes and engage in worker exploitation. Employers who violate prohibitions on unauthorized employment may be subject to civil monetary penalties and/or criminal penalties. Combatting Immigration Fraud There are two general types of immigration fraud: document fraud and benefit fraud. Some view immigration fraud as a continuum of events, because people may commit document fraud to engage in benefit fraud. The INA addresses immigration fraud in several ways. It makes "misrepresentation" (e.g., obtaining a visa by falsely representing a material fact or entering the United States by falsely claiming U.S. citizenship) a ground for inadmissibility. The INA also has civil enforcement provisions, distinct from removal or inadmissibility proceedings, to prosecute individuals and entities that engage in immigration document fraud. Apart from the INA, the U.S. Criminal Code classifies knowingly producing or using fraudulent immigration documents (e.g., visas, border crossing cards) as criminal offenses. In addition to relying on document inspection to determine if noncitizens are eligible for federal benefits, USCIS operates the Systematic Alien Verification for Entitlements (SAVE) system, which provides federal, state, and local government agencies access to data on immigration status. USCIS does not determine benefit eligibility; rather, SAVE enables program administrators to ensure that only those noncitizens and naturalized citizens who meet their own programs' eligibility rules actually receive public benefits. Unauthorized Aliens Determining how to address the unauthorized alien population residing in the United States has arguably been among the most divisive immigration issues facing Congress. Unauthorized aliens consist of those who (1) entered the country surreptitiously without inspection, (2) were admitted on the basis of fraudulent documents, or (3) overstayed their nonimmigrant visas. In all three instances, the aliens violated the INA and may be removed. Aliens who attempt to enter the United States illegally and those who assist them also are subject to INA-mandated penalties. Because the exact number of unauthorized aliens residing in the United States remains unknown, demographers have developed methods to estimate their population size and characteristics. According to the most recent and widely cited estimates available, the size of the unauthorized alien population increased from 8.6 million in 2000 to a peak of 12.2 million in 2007. It has fluctuated between 11.0 and 11.5 million since that time. Scholars attribute the general decline and changing country-of-origin composition in illegal migration flows in recent years to increased border security, relatively large numbers of alien removals, high U.S. unemployment, crime in Central America, and other factors. Options proposed for addressing the unauthorized alien population often emphasize reducing its size. Some approaches would require or encourage unauthorized aliens to depart the United States. Other strategies would grant qualifying unauthorized residents various immigration benefits, including an opportunity to obtain legal immigration status. Immigration Law Regarding Unauthorized Aliens Federal law places various restrictions on unauthorized aliens. In general, they have no legal right to live or work in the United States and are subject to removal from the country. At the same time, the INA provides limited forms of immigration relief for some unauthorized aliens to be legally admitted to the country, including crime victims or those seeking asylum. Unauthorized aliens who were present illegally in the United States for between 6 and 12 months are barred from readmission to the United States for 3 years, and those present for more than 1 year are barred for 10 years (the "3- and 10-year bars"). Deferred Action For unauthorized aliens who cannot obtain LPR status, existing mechanisms enable some to remain in the United States. One such mechanism, deferred action, is defined by DHS as "a discretionary determination to defer removal action of an individual as an act of prosecutorial discretion." Deferred action is not an immigration status and does not have a statutory authority; it is a form of administrative discretion. Examples of deferred action may include DHS terminating removal proceedings, declining to initiate removal proceedings, or declining to execute a final order of removal. Approval of deferred action means that no action will be taken against a removable alien for a specified time or in some cases indefinitely. Under the Deferred Action for Childhood Arrivals (DACA) initiative begun by DHS in June 2012, certain individuals without a lawful immigration status who were brought to the United States as children and who meet other criteria may be granted deferred action for two years, subject to renewal. DACA recipients can apply for employment authorization but are not afforded a pathway to a legal immigration status. DACA was initiated not by congressional legislation but by the Obama Administration. The Trump Administration announced the planned rescission of the DACA initiative on September 5, 2017, but due to federal court orders enjoining the rescission, DACA remains in effect pending the outcome of further litigation. | Summary: U.S. immigration policy is governed largely by the Immigration and Nationality Act (INA), which was first codified in 1952 and has been amended significantly several times since. At a fundamental level, U.S. immigration policy can be viewed as two sides of a coin. One side emphasizes the faciliation of migration flows into the United States according to principles of admission that are based upon national interest. These broad principles currently include family reunification, labor market contribution, humanitarian assistance, and origin-country diversity. The United States has long distinguished permanent immigration from temporary migration. Permanent immigration occurs through family and employer-sponsored categories, the diversity immigrant visa lottery, and refugee and asylee admissions. Temporary migration occurs through the admission of visitors for specific purposes and limited periods of time, and encompasses two dozen categories of visitors, including foreign tourists, students, temporary workers, and diplomats. The other side of the immigration policy coin emphasizes the restriction of entry to and removal of persons from the United States who lack authorization to reside in the country, are identified as criminal aliens, or whose presence in the United States is not considered to be in the national interest. Such immigration enforcement is broadly divided between border enforcement-at and between ports of entry-and other enforcement tasks including detention, removal, worksite enforcement, and combatting immigration fraud. The dual role of U.S. immigration policy creates challenges for balancing major policy priorities, such as ensuring national security, facilitating trade and commerce, protecting public safety, and fostering international cooperation. | 8,536 | 334 | gov_report | en |
Subsets and Splits
No saved queries yet
Save your SQL queries to embed, download, and access them later. Queries will appear here once saved.