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Summarize: By. Emily Crane For Daily Mail Australia. Australia's Great Barrier Reef is at risk because authorities are failing to protect the marine park by approving the dumping of dredge spoil, a former government official says. Jon Day, who was a former director of Conservation Biodiversity and World Heritage at the Great Barrier Reef Marine Park Authority, says the dumping of dredge spoil will put more pressure on a reef that is already in decline. The authority approved plans in January to dump three million cubic metres of dredge spoil at the Great Barrier Reef to expand Queensland's Abbot Point coal port. Scroll down for video. Australia's Great Barrier Reef is at risk because authorities are failing to protect the marine park by approving the dumping of dredge spoil from Abbot Point coal port, a former government official says. It's a decision that should not have been made, according to Mr Day, and is part of an ABC Four Corners investigation on Monday. Scientists and senior officials within the authority faced a tense year-long struggle against the proposal, with fears about the effect the spoil could have on the marine park. 'The big question is why was it allowed to be approved,' Four Corners reporter Marian Wilkinson told Daily Mail Australia. 'The official view from the Environment Minister (Greg Hunt) is that they've put tougher environmental conditions on so they believe it can be done safely. But there's a lot of questions among experts inside and outside the agency about it.' Jon Day, who was a former director of Conservation Biodiversity and World Heritage at the Great Barrier Reef Marine Park Authority, told ABC reporter Marian Wilkinson the dumping of dredge spoil would put more pressure on a reef that is already in decline. Mr Day, who resigned from the authority last month, says alternatives to sea dumping for Abbot Point weren't properly considered. 'If we take that into account and if we did a proper evaluation of all the alternatives, that decision would not have been made,' he said. 'Our own legislative mandate says "the long-term protection and conservation of the values", and we're not doing that.' Wilkinson said the decision was criticised by UNESCO's World Heritage Committee, which will decide next year whether the reef should be declared 'in danger'. The authority approved plans in January to dump three million cubic metres of dredge spoil at the Great Barrier Reef to expand Queensland's Abbot Point coal port. Reporter Marian Wilkinson Wilkinson said the decision was criticised by UNESCO's World Heritage Committee, which will decide next year whether the reef should be declared 'in danger' 'The other options to look at were trying to find a place to dump on land and trying to do something to extend the port's trestles so that ships didn't have to come so close to the reef,' Wilkinson said. Mr Hunt, the federal environment minister who approved the dumping, told the program that Abbot Point was a 'line in the sand' and he has guaranteed that no further dumping will take place in the marine park under his watch. 'Of course, the argument from some of the scientists is why did we have to have this one in the first place?' Wilkinson said. The ABC's Four Corners is on Monday night at 8.30pm. Scientists and senior officials within the authority faced a tense year-long struggle against the proposal, with fears about the effect the spoil could have on the marine park | Summary: A senior official at the Great Barrier Reef Marine Park Authority says dumping more dredge would put extra pressure on the reef. The authority approved plans in January to dump 3 million cubic metres of dredge spoil at the reef to expand Queensland's Abbot Point coal port. It's a decision that shouldn't have been made, according to scientists. Federal Environment Minister Greg Hunt has vowed to stop future dredge spoil at the marine park. | 756 | 98 | cnn_dailymail | en |
Summarize: By. Jennifer Newton. Helicopters are used to airlifting precious cargo to safety but nothing could have prepared the crew for transporting this heavy load. A four tonne white rhino needed to be transferred to a different enclosure in the Zululand region of Kwa-Zulu Natal in South Africa so the animal was airlifted using a harness. Em Gatland captured the moment the rhino was lifted up by the helicopter in a programme by KZN Wildlife Ezemvelo Game Capture. The white rhino was being transferred to a different enclosure in the Zululand region of Kwa-Zulu Natal in South Africa. Conservationists relocate pairs of rhinos every year in the hope of ensuring the strength of future blood lines in the animals. The location of their new home has not been revealed so the animals can be kept safe. The photographer said: 'Taking pictures of the rhino was a surreal feeling and a mixture of emotion. 'With the current rhino crisis, to be so up close and personal to the prehistoric creature was a breath-taking experience.' Rhinos in that area of South Africa are relocated in pairs every year for the benefit of the species and in order to ensure the strength of future bloodlines. But the location they are moved to is kept secret, to keep the animals safe. After sedating the animal, conservationist prepare for the airlift by securing it in a harness. The helicopter flies in, ready to take the rhino to a new enclosure, which remains secret to protect the animal's safety. Conservationists prepare to catch the rhino as it comes into land in its new enclosure in South Africa. The rhino was first darted with anaesthetic in order for the animal conservationists to undertake the mammoth task of securing the rhino so it could be safely airlifted to its new home. Ms Gatland added: 'Airlifting the rhino was a big team operation and the biggest difficulty for me was manoeuvring in-between people to get a good picture of the rhino. 'I didn't want to get in the way of the people working to get the massive mammal airlifted but I'm glad I managed to get some good shots. 'It was a very moving and emotional moment and a completely new experience for me. To be up so close and personal to a prehistoric creature is a breath taking experience. The four tonne rhino begins to rouse from the sedation after being transported to its new home. The rhino will now be closely monitored for five weeks to make sure it is happy in its new environment. 'I became emotionally aware of how vulnerable they really are. It was a humbling experience to say the least and I couldn't but feel a completely overwhelming desperation for the precious creature. 'There is an element of danger as I was in a Big 5 reserve in complete wilderness and I needed to have my wits about me. 'It really gave me a feeling of perspective in this world.' The rhino will now be closely monitored for five weeks to make sure it is happy in its new environment | Summary: The rhino, who weighs four tonnes, was airlifted to a new enclosure in Zululand, Kwa-Zulu Natal in South Africa. Conservationists relocate the animals in pairs annually to ensure the strength of their bloodlines. Images of the dramatic relocation were taken by photographer Em Gatland, who described the experience as'surreal' Animal will now be monitored for five weeks to check it is happy in its new enclosure. | 675 | 97 | cnn_dailymail | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Ephedrine Alkaloids Regulation Act of 2005''. SEC. 2. FINDINGS. The Congress finds as follows: (1) The United States faces increasing danger related to methamphetamine trafficking, production, and abuse. (2) Methamphetamine is a highly addictive drug that can be readily made from products and precursors purchased from retail stores. Step-by-step recipes can easily be found on the Internet, which is a factor in the dramatic increase in the number of clandestine labs in recent years. (3) Methamphetamine-producing clandestine laboratories have been identified by the Drug Enforcement Administration as a significant threat to the Nation's public health and safety. The manufacture of methamphetamine produces highly toxic and unstable chemicals that threaten the well-being of first responders, law enforcement officers, and the community at- large. (4) Methamphetamine production, once exclusively found in West Coast States, has rapidly moved eastward to the Midwest. Production can now be found on the East Coast, in the States of New York and Florida. (5) Methamphetamine abuse is indiscriminate of age, socioeconomic level, or race. (6) Pseudoephedrine is a necessary precursor chemical in the production of methamphetamine, which prompted the Drug Enforcement Administration to initiate investigations regarding the chemical's sale and distribution. (7) Efforts to reduce access to pseudoephedrine by methamphetamine producers, such as blister packaging and sales thresholds, have not been effective deterrents, and pseudoephedrine tablets remain pervasive in the illicit production of methamphetamine. (8) Pseudoephedrine in liquid gel and liquid forms have not been found to be used in methamphetamine production. (9) As States and communities attempt to combat and control methamphetamine through restricting the sale of pseudoephedrine products, it is incumbent upon the Congress to develop a uniform standard for the distribution of pseudoephedrine in tablet form. SEC. 2. CONTROLLED SUBSTANCES; ADDITION OF EPHEDRINE ALKALOIDS AND PHENYLPROPANOLAMINE TO SCHEDULE V. (a) In General.--Effective upon the expiration of 30 days after the date of the enactment of this Act, ephedrine alkaloids (including ephedrine and pseudoephedrine) and phenylpropanolamine, and their salts, optical isomers, and salts of optical isomers, whether alone or in combination with other substances, shall be considered to be listed in schedule V of the schedules of controlled substances established under section 202(c) of the Controlled Substances Act, subject to subsection (b) and subject to the authority of the Attorney General under such Act to designate substances as controlled substances or listed chemicals. The Attorney General shall amend part 1308 of title 21, Code of Federal Regulations, accordingly. (b) Certain Forms of Pseudoephedrine and Phenylpropanolamine.-- Subject to the authority of the Attorney General under the Controlled Substances Act to designate substances as controlled substances or listed chemicals-- (1) subsection (a) does not apply to pseudoephedrine or phenylpropanolamine when contained in a drug that is in liquid or gel form and is marketed or distributed lawfully in the United States under the Federal Food, Drug, and Cosmetic Act; and (2) pseudoephedrine or phenylpropanolamine when so contained shall be considered a listed chemical. SEC. 3. REGULATION OF TRANSACTIONS INVOLVING LISTED CHEMICALS; EXEMPTION FOR CERTAIN DOSAGE FORMS AND QUANTITIES OF PSEUDOEPHEDRINE OR PHENYLPROPANOLAMINE. (a) Definition of Regulated Transaction.--Section 102(39)(A)(iv) of the Controlled Substances Act (21 U.S.C. 802(39)(A)(iv)) is amended-- (1) in the matter preceding subclause (I), by striking ``unless--'' and inserting ``unless, subject to clause (v)--''; and (2) in subclause (II)-- (A) by inserting ``in liquid or gel form'' after ``containing pseudoephedrine or phenylpropanolamine products''; and (B) by striking ``shall be 9 grams of pseudoephedrine or 9 grams of phenylpropanolamine'' and inserting ``shall be any quantity in excess of 9.0 grams of pseudoephedrine or 9.0 grams of phenylpropanolamine''. (b) Definition of Ordinary Over-the-Counter Pseudoephedrine or Phenylpropanolamine Product.--Section 102 of the Controlled Substances Act (21 U.S.C. 802) is amended-- (1) in paragraph (39)(A)(iv)(I)(aa), by striking ``, except that'' and all that follows through ``1996)''; and (2)(A) by striking paragraph (45); and (B) by redesignating paragraph (46) as paragraph (45). (c) Chemical Mixtures not Easily Used in Illicit Production.-- Section 102(39)(A)(v) of the Controlled Substances Act (21 U.S.C. 802(39)(A)(v)) is amended by inserting after ``chemical mixture'' the following: ``(including a mixture that may be marketed or distributed lawfully in the United States under the Federal Food, Drug, and Cosmetic Act)''. | Title: To amend the Controlled Substances Act with respect to the regulation of ephedrine alkaloids, including ephedrine and pseudoephedrine, and for other purposes Summary: Ephedrine Alkaloids Regulation Act of 2005 - Requires ephedrine alkaloids (including ephedrine and pseudoephedrine) and phenylpropanolamine to be listed in schedule V of the Controlled Substances Act (CSA) (drugs or other substances that have a low potential for abuse and that have a currently accepted medical use in treatment in the United States, abuse of which may lead to limited physical or psychological dependence). Excepts pseudoephedrine and phenylpropanolamine when contained in a drug that is in liquid or gel form marketed or distributed lawfully in the United States under the Federal Food, Drug, and Cosmetic Act (FDCA), which shall be considered a listed chemical. Makes such requirement and exception subject to the Attorney General's authority to designate substances as controlled or listed chemicals. Amends CSA to revise the definition of "regulated transaction" to: (1) provide that the threshold for any distributor sale of products containing pseudoephedrine products in liquid or gel form, or containing phenylpropanolamine products, shall be nine grams of pseudoephedrine or phenylpropanolamine; and (2) specify as a "chemical mixture" a transaction in which is excluded from such definition a mixture that may be marketed or distributed lawfully in the United States under FDCA. | 1,321 | 327 | billsum | en |
Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a baseball pitching machine, intended for use as a substitute for a human pitcher during baseball batting practice and adapted to pitch both straight and curve balls to a batter so that the ball enters the strike zone at substantially the same spot during each repetition of a series of throws. 2. Description of the Prior Art There have been many attempts to obtain uniform control of mechanically thrown baseballs. Ball throwing devices employing a swinging arm, a mechanical impact means or spring loaded propelling devices are notorious for instability and inaccuracy. In addition, such machines cannot produce the variety of pitches that a batter is likely to encounter on the playing field. In recent years, pitching machines have been produced which use two oppositely rotating wheels positioned so that a nip is created into which a baseball may be introduced from one side for projection from the other side. An example of such a machine is that of Doeg (U.S. Pat. No. 3,604,409 issued Sept. 14, 1972). Another example of such a machine is Halstead (U.S. Pat. No. Re. 28,462, reissued July 1, 1975). The baseballs delivered by a machine such as the Doeg machine and the Halstead machine are not consistently thrown over many repetitions into the same spot or substantially the same spot in the batter's strike zone. The consistency of these machines is adversely affected by the use of a separate motor for each wheel. The inevitable slight perturbation in the speed of an electric motor causes the relative angular velocity of wheels driven by two such motors to vary over an impermissibly wide range. Such variations in relative angular velocity cause the spin imparted to the ball to change at random from one throw to the next. The random nature of such changes makes the strike zone arrival point of the ball quite unpredictable. If the batter cannot relay on the balls arriving at substantially the same spot during a series of ball projections, he is not able to concentrate upon hitting the particular type of pitch for which he is preparing. Improved predictability would be particularly advantageous in the training of younger batters who fear being hit by the ball. The young batter's confidence level is increased in direct proportion to an increase in ball arrival predictability. Accordingly, it is an object of this invention to provide a baseball pitching machine able to deliver a curve ball into substantially the same spot of the batter's strike zone at each repetition of a series of practice pitches. Another object is to provide a baseball pitching machine of the counter-rotating wheel pair type in which both wheels are turned by means of one electric motor so that fluctuations in the speed of such is motor damped out or cancelled by the distribution of such fluctuations over both wheels of the pair of counter-rotating wheels. SUMMARY OF THE INVENTION The baseball pitching machine of this invention includes two oppositely rotating wheels, the axles of which are held in rotational relationship with a horizontal platform by means of bearings, the axle of one wheel engages a variable drive pulley below the horizontal platform, and a standard pulley on the other axle engages the drive pulley of an electric motor. A double V-belt connects all of the pulleys. Tension on the V-belt is adjusted by means of a tensioner pulley mounted on a block sliding in a channel and controlled by a crank. As the tension on the V-belt is changed at the tension pulley, the V-belt is caused to move more or less closely to the center of the variable drive pulley. In operation, the speed of the wheel attached to the variable drive pulley is controlled by turning the crank attached to the sliding block on which the tensioner pulley is mounted. The motor, wheels, and pulleys of the present invention are mounted in a multi-sectional frame appropriately jointed and with pivots to allow adjustments to be made in the elevation and direction of the path of the projected ball. At the bottom of the frame are roadwheels at one end and a wagontongue at the other to facilitate movement of the machine onto and off of the playing field. The motor is a DC shunt wound motor which is connected to a standard 60 cycle 120 volt AC power supply through an appropriate rectifier. The field voltage of the motor is controlled by means of a suitable rheostat. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view showing a baseball pitching machine embodying the present invention; FIG. 2 is a sectional view taken along line 2 showing elevation changing means; FIG. 3 is a sectional view taken along line 3 showing direction changing means; and FIG. 4 is an exploded isometric sectional detail of the relative opposite rotational angular velocity changing means. DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to the figures wherein numerals designate like parts, a preferred embodiment of the invention will be described in detail. With reference to FIG. 1, one embodiment of a baseball pitching machine is shown wherein oppositely rotating wheels 12 and 14 stand on axes 16 and 18 so that when ball 105 rolls down track 106 it is caught in the nip between wheels 12 and 14 and projected along the path indicated by the arrows. Wheels 12 and 14 are controlled by certain electro-mechanical devices contained between plates 22 and 36 which will be described in detail in conjunction with the explanation of FIG. 4. Plates 22 and 36 are joined together with spacer posts 102 through which pass nut and bolt combinations 104. Thus joined, plates 22 and 36 form a single unitary platform capable of swinging upward around pivot 120. Such upward pivoting is controlled by hand wheel 112 which is affixed to jackscrew 114. The operation of jackscrew 114 will be described in more detail in conjunction with the explanation of FIG. 2. Pivot hinges 120 and jackscrew 114 connect plate 36 with middle frame platform 108. Middle frame 108 sits atop lower Y-shaped frame 110. Middle frame 108 swivels with respect to lower Y-shaped frame 110 under the control of hand wheel 122 connected to jackscrew 124 turning within tapped-block 125. The manner by which middle frame 108 is made to swivel is described in more detail with reference to FIG. 3 below. A bridge rectifier 158 of a conventional design is also mounted on middle frame 108. Lower Y-shaped frame 110 is connected to ground wheel 132 and axle 134 by means of suspension bar 136. At the end of the Y-shaped frame opposite axle 134 is mounted ground plate 138. Ground plate 138 pivots by means of brackets 140 and pins 142 at the lower end of spacer bars 144. Spacer bars 144 are drilled at selective spacings 145 so that the wagontongue formed by lower Y-shaped frame 110 can be positioned at selective distances above the ground. Such positioning of the wagontongue is accomplished by sliding spacer bars 144 within brackets 146 attached by means of bolts 148 to wagontongue mounting plate 149. Spacings in bar 144 are selected by means of pins 150. Also mounted on the wagontongue end of lower Y-shaped frame 110 is control rheostat 152, swivel index strip 130, and tapped-block 125. Swivel index marker 128 is affixed to fuse box 154 which, in turn, is attached to middle frame 108. Now referring to FIG. 2, vertical trajectory changing means will be described. Phantom lines in FIG. 2 depict the change in position of plates 36 and 22 made possible by the cooperation of hinge 120 and jackscrew 114. As may be seen by reference to FIG. 2, plate 36 carries motor 62 with its support flange 65 and drive pulley 68. Plate 36 also carries idler pulley 70 mounted on idler pulley block 76. Mounted through plates 22 and 36 is the axle 16 of wheel 12 held in place by bearings 20 and 58. On axle 16 may be seen wheel drive pulley 56. Plate 36 is forced upward against the weight of the aforesaid supported elements by the pressure of jackscrew 114 against foot 118 when turned by handcrank 112 so that jackscrew 114 is driven through tapped-block 116. Such pressure by jackscrew 114 causes plate 136 to pivot on pivot hinge 120. Pivot hinge 120 and tapped-block 116 are both mounted on middle frame 108 as shown in FIG. 2. Middle frame 108 swivels in relation to lower Y-shaped frame 110 by means of swivel pin 126. The means by which middle frame 108 is swivelled is described in more detail by reference to FIG. 3. Referring now to FIG. 3, the positions which may be assumed by swivelling middle frame 108 are shown in phantom. Swivel pin 126 is shown mounted on lower Y-shaped frame 110. Hand wheel 122 turns jackscrew 124 through tapped-block 125 (not shown). The end of jackscrew 124 opposite hand wheel 122 is nested in swivel foot 123 (not shown). Swivel foot 123 is affixed to middle frame 108 so that when it is pulled towards one side or the other of lower Y-shaped frame 110, middle frame 108 attached to it is made to swivel about swivel pin 126. With reference now to FIG. 4, a means of varying the relative angular velocity of the oppositely rotating wheels is described in detail. FIG. 4 is a detail showing plates 22 and 36 along with associated hardware. Plate 22 is cut away to reveal a system of pulleys. Axle 18 is shown passing through plate 22 and bearing 24. Bearing 24 is held against plate 30 by means of bolts 28. Mounting plate 30 is held against plate 22 by bolts 26. When bolts 26 are loosened, bolts 26 are allowed to slide in slots 32 which have been cut through plate 22. Axle 18 is axially attached to variable drive pulley 34 and passes through variable drive pulley 34 terminating in bearing 46 below plate 36. Bearing 46 is attached to mounting plate 42 by means of nuts 48 and bolts 50. The mounting plate in turn is affixed to the lower side of platform 36 by means of nuts 38 and bolts 40. When nuts 38 are loosened, bolts 40 are free to slide within slots 44 which have been cut into plate 36. This arrangement is the same as that described in conjunction with bearing 24. Axle 16 passes through plate 22 and bearing 20. Bearing 20 is held against plate 22 by means of nuts 54 and bolts 52. Bearing 58 is attached to plate 36 by bolt 60. Between bearings 58 and 20, axle 16 is attached to wheel drive pulley 56. Motor 62 is secured to plate 36 by bolting its flange 65 between spacer posts 64 and bolts 66 passing through spacer posts 64 and secured to plate 36. Motor 62 turns drive pulley 68. Double V-belt 69 grips drive pulley 68, wheel drive pulley 56, and idler pulley 70. Idler pulley 70 is secured to plate 36 at bearing block 72. Bearing block 72 is drawn towards tapped-block 74 by thumbscrew 76. Double V-belt 69 passes from idler pulley 70 to idler pulley 78. Idler pulley 78 rotates on bearing block 80 which is bolted to plate 36 by means of bolt and nut combination 86. When bolt and nut combination 86 is loosened, bearing block 80 may slide back and forth in slots 87 cut into plate 36. The sliding of bearing block 80 when nut and bolt combination is loosened is controlled by means of tapped-block 82 and thumbscrews 84. Thumbscrews 84 press bearing block 80 so that idler pulley 78 is urged against the tension of double V-belt 69. From idler pulley 78, double V-belt 69 passes around variable drive pulley 34 to idler pulley 88. Idler pulley 88 turns upon bearing block 92. Bearing block 92 slides within a channel created by U-shaped block 90 bolted to plate 36. The channel formed by retainer block 90 is substantially in the shape of an inverted Y. Bearing block 92 is machined to conform to the channel of retainer block 90. The position of bearing block 92 within its channel is controlled by means of hand wheel 94 turning jackscrew 95 through tapped-block 96 and terminating at a foot machined into bearing block 92. Setscrew 98 may be tightened to prevent slippage in jackscrew 95. In operation, drive pulley 68 turns in a counter-clockwise direction while pulley 56 turns in a clockwise direction. Idler pulley 70 turns in a counter-clockwise direction while idler pulley 78 turns in a clockwise direction. Variable drive pulley 34 turns in a counter-clockwise direction and idler pulley 88 turns in a clockwise direction. Force is transmitted from motor drive pulley 68 to pulley 56, 70, 78, 34, and 88 by means of a double faced V-belt. Idler pulley 88 in cooperation with jackscrew 95 functions as a means of changing the rotational speed of axle 18. As idler pulley 88 is forced against the tension of double faced V-belt 69 by operation of hand wheel 94, the tension of double faced V-belt 69 is increased causing a change in the spacing of spring-loaded variable speed pulley 34. As the tension of double faced V-belt 69 and the biasing of spring-loaded variable drive pulley 34 counteract each other, an equilibrium is reached. The new equilibrium will correspond to a position of the double faced V-belt 69 which is closer to axle 18 causing axle 18 to turn faster in a counter-clockwise direction. When the pressure of idler pulley 88 against double faced V-belt 69 is eased by counter-clockwise rotation of hand wheel 94, the tension on double faced V-belt 69 is relaxed. A relaxation of tension in double faced V-belt 69 enables spring-loaded variable drive pulley 34 to force double faced V-belt 69 away from axle 18 with a consequent slowing of the counter-clockwise rotational speed of axle 18. Motor 62 is a one-third horsepower DC variable speed motor. One hundred and twenty volt AC current is received and rectified by conventional circuitry contained in box 158 of FIG. 1. DC current is then applied to rheostat 152 where an appropriate DC voltage is selected. The voltage at rheostat 152 is selected to correspond to a desired forward ball speed. Hand wheel 94 is then adjusted to select the desired difference in RPMs between wheel 12 and wheel 14. It is this difference in RPMs which imparts the spin to the baseball. By adjusting the RPM difference by means of hand wheel 94, various types of curve, slider, and knuckleballs are projected by the machine. When the desired ball behavior has been attained, setscrew 98 is tightened against further rotation of hand wheel 94. The machine will now deliver a baseball to substantially the same spot in the batter's strike zone with a trajectory that is consistent over many repetitions. In order to accommodate batters of differing heights and in order to change the strike zone entry point without altering the spin behavior of the ball, hand wheels 112 and 122 may be adjusted as appropriate. While a variety of pulleys may be used in conjunction with this invention, the present embodiment employs a Wood's FHP spring-loaded sheave number 6600 having a height of three and seven-eighths inches and diameter of six inches for variable drive pulley 34. In the present embodiment, idler pulleys 70, 78, and 88 are Fafnir brand pulleys number 010-10874 of diameter four inches and width seven-eighths of an inch. Bearings 24 and 20 are Fafnir brand bearing number RA100RRB having a height of 1.343 inches and an outer bore edge diameter of one and one-half inches. The machine of the present invention can be fully calibrated after only five ball pitching repetitions. Its shunt wound DC motor is especially appropriate due to its constant speed characteristics. Although a preferred embodiment of the invention has been described in detail, it is to be understood that various changes and substitutions can be made without departing from the spirit and scope of the invention as defined by the appended claims. | Summary: A baseball-pitching machine wherein a baseball is delivered into the constricted space between, and thereby gripped frictionally by, to oppositely rotating wheels which throw the ball. A single DC shunt wound motor is used to drive the wheels in cooperation with one variable drive pulley and an assortment of guide pulleys. One wheel is driven at a constant speed by the motor while the speed of the second wheel is adjusted by means of a variable drive pulley. By thus changing the speed of one of the two oppositely rotating wheels, it is possible to impart a variety of spins to the thrown ball and thus simulate curve and slider balls thrown by a professional pitcher. The axis of the variable drive pulley is fixed and the position of the belt within the variable drive pulley is controlled indirectly by means of a belt tensioning pulley operated by a screw. | 4,254 | 191 | big_patent | en |
Summarize: Shocking images depicting U.S. soldiers burning the bodies of what appear to be Iraqi insurgents, have emerged today. The explosive photographs, reportedly taken in Fallujah in 2004, have already sparked a Marine Corps investigation, but many of the 41 gag-inducing shots are just too grisly to publish. Two pictures show a Marine pouring what looks like gasoline on the remains of enemy soldiers and another two images appear to show the remains go up in flames. Two more capture the horrifically charred bodies. WARNING: EXTREMELY GRAPHIC CONTENT. Horrific: Shocking images depicting U.S. soldiers burning the bodies of what appear to be Iraqi insurgents, have emerged today. Burning: The explosive photographs, reportedly taken in Fallujah in 2004, appear to show U.S. soldier pouring gasoline on the bodies of Iraqi insurgents. The sick snaps were exclusively obtained by TMZ, who turned them over to the Pentagon last week, triggering the probe. According to the website, U.S. Central Command, which is in charge of military operations in the Middle East, reviewed the photos to determine if they had been brought to their attention before. They determined they had not. Other horrific pictures show a Marine squatting next to a skull to pose for the camera. His U.S. military uniform is clear, on his face he wears a wide grin and he is pointing his gun at the skeleton. Another picture shows a soldier rifling through the pockets of the scant remains of an Iraqi soldier. TMZ. said it has withheld the bulk of the images - including one showing a. body being eaten by a dog - because they are just too graphic. Grim: Many of the 41 gag-inducing shots are just too grisly to publish. probe: The gruesome images have already sparked a Marine Corps investigation. Charred: Two more pictures capture the horrifically charred bodies. It reported seeing well over a dozen dead insurgents in total in the heinous pictures, in various states, including some covered in flies. The Department of Defense said the pictures appear to show U.S. soldiers in violation of the Uniform Code of Military Justice. The code outlines that it is a crime to mishandle remains. There is no statute of limitations on. the crime, which means the Marines can be prosecuted even if they're no. longer active in the military. If convicted, the soldiers could go to. prison. 'We are aware of photos appearing on. TMZ.com that depict individuals in U.S. Marine uniforms burning what. appear to be human remains,' Cmdr Bill Speaks, from the Secretary of Defense's office, told MailOnline Wednesday. Pentagon: The sick snaps were exclusively obtained by TMZ, who turned them over to the Pentagon last week, triggering the probe. Posing: Other horrific pictures show a Marine squatting next to a skull to pose for the camera. His U.S. military uniform is clear, on his face he wears a wide grin and he is pointing his gun at the skeleton. Pickpocket: The Department of Defense said the pictures appear to show U.S. soldiers in violation of the Uniform Code of Military Justice. The code outlines that it is a crime to mishandle remains. 'The Marine Corps is currently investigating the veracity of these photos, circumstances involved, and if possible, the identities of the service members involved. 'The findings from this investigation will determine whether we are able to move forward with any investigation into possible wrongdoing.' Some have suggested the Marines may have been burning the remains as a sanitary measure. However,. Pentagon spokesman Army Col. Steven Warren said the proper handling of. war remains is set by U.S. military regulation and that the actions. depicted in the photos 'are not what we expect from our service. members.' Cmdr Speaks said the deplorable acts depicted in the images are not representative of the millions of hardworking men and women who have served in the Middle East. 'The actions depicted in these photos are not what we expect from our service members, nor do they represent the honorable and professional service of the more than 2.5 million Americans who have served in Iraq and Afghanistan,' he told MailOnline. In 2005 report, U.S. soldiers in Gumbad, Afghanistan were investigated for burning the bodies of two enemy fighters. The men argued they set alight the corpses for hygienic reasons, after local citizens had not retrieved the bodies after 24 hours. A report concluded that the action indicated poor judgement but was not a war crime. It stated: 'Based on the criminal investigation, there was no evidence to substantiate the allegation of desecration or any violation of the Law of War. However, there was evidence of poor decision-making and judgment, poor reporting and lack of knowledge and respect for local Afghan customs and tradition.' | Summary: The explosive photographs, reportedly taken in Fallujah in 2004, have sparked a Marine Corps investigation. However, many of the 41 shots, obtained by TMZ, are just too grisly to publish. Two pictures show a Marine pouring gasoline on the enemy remains, another two images show the Iraqi soldiers going up in flames while a fifth picture captures the charred bodies. U.S. Central Command, which oversees military operations in the Middle East, determined the photos had not been brought to their attention before. | 1,117 | 122 | cnn_dailymail | en |
Summarize: This divisional application claims priority under 35 U.S.C. § 120 from co-pending U.S. patent application Ser. No. 10/978,897 filed on Nov. 1, 2004 by David N. Heinsey et al. with the same title, the full disclosure of which is hereby incorporated by reference. TECHNICAL FIELD This invention relates generally to a system and method for detecting existence of a failed or broken shear bolt supporting all or a portion of a concave of a threshing system of an agricultural combine and, more particularly, to a system and method which utilizes information relating to rates of movement and absence of movement of a concave to determine the existence and nonexistence of a failed or broken shear bolt. BACKGROUND ART Commonly, one or more shear bolts are utilized in support of a concave or a section of a concave extending partially around a bottom portion of a rotor of a threshing system of an agricultural combine, which shear bolt or bolts are designed to fail or break to allow the concave or concave section to fall away from the rotor when large slugs of crop material and/or hard foreign objects enter the space between the concave segment and the rotor. This is intended to prevent damage to the threshing system, but also results in degraded performance of the threshing system Typically, if a shear bolt breaks to allow a segment of the concave to fall away from the rotor, contamination in the clean grain and/or discharge of larger pieces of crop material from the crop residue system of the combine will be noticed. Often, the investigation into the decreased performance will begin or will be concentrated on the cleaning system of the combine, such that excessive machine downtime may be required before the failed concave shear bolt is discovered. Thus, what is sought is a manner of detecting a failed or broken concave shear bolt automatically and quickly, and which is simple and economical to implement. SUMMARY OF THE INVENTION What is disclosed is a system and method for detecting a failed or broken shear bolt supporting a concave of a threshing system of an agricultural combine, which provides one or more of the sought after benefits set forth above. According to a preferred aspect of the invention, the threshing system includes a rotatable rotor and at least one concave segment extending around a lower region of the rotor in spaced relation thereto. One longitudinally extending edge of the concave segment is preferable pivotally or hingedly supported to allow movement of the concave segment upwardly and downwardly in relation to the rotor. Such upward and downward movement is preferably accomplished by a driver, which can be, for instance, but is not limited to, a rotary or linear electric motor or actuator, a fluid cylinder, or the like, connected to the concave by a linkage including a shear bolt designed to fail or break when a force is applied against the concave urging it away from the rotor and of a sufficient magnitude to potentially damage the rotor and/or concave. The system also preferably includes a device or sensor such as, but not limited to, a potentiometer or Hall Effect sensor, for sensing or determining a position of the concave relative to the rotor or another suitable location. According to a preferred method of operation of the system, the position of the concave is monitored and, if a rate of change of the position in a downward direction exceeds a predetermined value, it is determined that the shear bolt is failed or broken, and a signal representative thereof is outputted. If the concave is at or near its lowest position when the driver is operated to raise the concave, the position thereof will be monitored and, if the position does not change accordingly, it will be determined that the shear bolt is broken. Additionally, if the shear bolt is indicated as being broken and the drive is operated and corresponding movement of the concave is determined, the broken shear bolt condition will be determined to be false. As a result, both the existence and absence of a failed or broken shear bolt can be determined according to the system and method of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a simplified schematic representation of a rotor and concave of a threshing system of an agricultural combine, showing elements of a system including a representative driver controllably operable for moving the concave relative to the rotor, and a linkage including an intact shear bolt connecting the driver to the concave, the system being operable for detecting failure or breakage of the shear bolt according to the invention; FIG. 2 is another simplified schematic representation of the rotor and concave of FIG. 1, showing the shear bolt broken to disconnect the driver from the concave and allow the concave to fall away from the rotor; FIG. 3 is a high level flow diagram of steps of the method of operation of the system for detecting failure or breakage of the shear bolt of FIGS. 1 and 2 ; FIG. 4 is another high level flow diagram of steps of the method of operation of the system for detecting failure or breakage of the shear bolt of FIGS. 1 and 2 ; FIG. 5 is still another high level flow diagram of steps of the method of operation of the system for detecting failure or breakage of the shear bolt of FIGS. 1 and 2 ; and FIG. 6 is another high level flow diagram of steps of a method of operation of a system for detecting failure or breakage of the shear bolt of FIGS. 1 and 2. DETAILED DESCRIPTION OF THE INVENTION Referring now to the drawings, in FIGS. 1 and 2, representative threshing apparatus 10 of a threshing system of an agricultural combine is shown. Threshing apparatus 10 includes a cylindrical rotor 12 rotatably driven about a central longitudinal axis 14 therethrough. Threshing apparatus 10 also includes at least one semi-cylindrical shaped concave 16 positioned so as to extend around a lower region of rotor 12 of all or a segment of the length thereof. Here, it should be recognized or understood that concave 16 is intended to be representative of a concave that extends longitudinally along only a portion of the length of rotor 12, or along the entire length thereof. Concave 16 is shown supported along one longitudinally extending edge thereof, by a pin 18, for pivotal movement relative to rotor 12, as generally denoted by arrows A. In this regard, in FIG. 1, concave 16 is shown in what would be considered an operative position in spaced relation to an outer cylindrical surface of rotor 12, for threshing and separating grain introduced with other crop material in the space between rotor 12 and concave 16. The separated grain would then pass through holes or perforations in the surface of concave 16 so as to subsequently fall or be conveyed into a cleaning system (not shown) of the combine for further processing in the well known manner. The opposite longitudinally extending edge of concave 16 is supported by a linkage assembly 20 including a link arm 22 supported for rotation on a pin 24 connected to a frame or other structural member of the combine for rotation thereabout, as denoted by arrows B, a distal end 26 of link arm 22 being pivotally connected by a bolt 28 to a link 30 which, in turn, is connected by a shear bolt 32, to concave 16. Link arm 22 is additionally connected to a driver 34 including a rod 36 extendable upwardly, and retractable downwardly, as denoted by arrows C, for rotating distal end 26 of link arm 22 upwardly and downwardly, as denoted by arrows B, for raising and lowering link 30 and concave 16 as denoted by arrows A. Here, it should be noted and understood that driver 34 is representative of a wide variety of drivers and actuators that could be used in connection with concave 16 for raising and lowering it to achieve a desired spacing in relation to rotor 12, which drivers and actuators can include, but are not limited to, electric rotary and linear motors or actuators, fluid cylinders and the like. It should also be understood that linkage assembly 20 is but an example of a wide variety of different linkage assemblies and arrangements and other apparatus that can be used in connection between a driver, such as driver 34, and concave 16 for effecting movement of concave 16. Referring more particularly to FIG. 1, driver 34 is controllably operable by a control system 38 preferably including a suitable controller 40 such as a conventional processor based controller including a memory 42 and connected by one or more conductive paths 44 to driver 34 and a signal or display device 46, and also to a position sensor 48 associated with concave 16 for determining a position thereof relative to rotor 12 or another location and outputting information representative thereof to controller 40. Here, position sensor 48 is in connection with pin 18 so as to be operable for determining a pivotable or rotational position of concave 16 about an axis of rotation of pin 18, although it should be understood that a wide variety of other sensor devices, such as a proximity sensor or the like, could be used for determining the position of concave 16. More particularly in this regard, position sensor 48 can be a commercially available and conventionally operable potentiometer or Hall Effect sensor, as just two examples. As noted previously, in FIG. 1, concave 16 is shown at an operative position in a selected spaced relation to rotor 12, for separating grain from other crop material introduced into the space by the rotation of rotor 12 in the well known manner. In contrast, in FIG. 2, concave 16 is shown dropped or fallen from link 30 of linkage assembly 20 to a non or less operative position, as a result of failure or breakage of shear bolt 32 in such a manner so as to cause disconnection of link 30 from concave 16. Here, by the term “failure”, what is meant is a shearing or other breakage of shear bolt 32 in such a manner that concave 16 is disconnected or disengaged from link 30, so as to be capable of freely falling downwardly away from rotor 12 to thereby enlarge the space therebetween. This will typically occur as a result of induction or passage of a large slug or slugs of dense crop material into the space between rotor 12 and concave 16, or the induction of hard foreign objects into the space, which, at least partially as a result of the rotation of rotor 12, will apply a radially outwardly directed force against concave 16, which will be translated thereby and concentrated against the one or more shear bolts 32, which will have a predetermined load carrying capability. Thus, if the force applied against concave 16 and translated to the one or more shear bolts 32 exceeds the design limit of the shear bolt 32, the shear bolt will fail or break, thus releasing concave 16 to fall away from rotor 12, in the well known manner. It has been observed that if a shear bolt 32 is broken by application of a force thereagainst exceeding the load limit thereof, the applied force can cause concave 16 to rapidly or abruptly fall away from rotor 12, so as to result in a rate of change in the position of concave 16 which will be greater than that which will typically occur as a result of normal movements of concave 16 by driver 34. Information representative of such rapid rate of change will be outputted by position sensor 48 to controller 40, which can be programmed to compare the sensed rate of change to one or more stored values which can be representative of, for instance, a maximum rate of normal downward movement of concave 16 by driver 34. As a result, if the sensed rate of positional change exceeds the stored value, controller 40 can be programmed to determine that a broken shear bolt condition exists. Controller 40 can then store information representative of this condition in memory 42 and, if desired, output a warning or alarm signal to a display, such as display 46, and/or to a warning alarm or the like for alerting the combine operator or other personnel. As another aspect of the invention, if concave 16 is at a lower extreme or limit of its travel relative to rotor 12 and breakage of shear bolt 32 occurs, the rapid falling of concave 16 may not occur. However, subsequently, when driver 34 is operated for raising concave 16, if no corresponding raising or change in position of concave 16 is sensed by position sensor 48, for instance, for a specified period of time, controller 40 can be programmed to determine that a broken shear bolt condition exists and store information representative thereof and/or output a signal or alarm representative thereof, as desired. Still further, if shear bolt 32 has been previously broken and repaired, or erroneously found to have been broken, controller 40 can operate driver 34 to move concave 16 and, if a resultant positional change is detected by position sensor 48, controller 40 can be programmed to determine that shear bolt 32 is intact or functional, and store information representative of that condition in memory 42 and/or output a signal representative thereof or cancel a signal or alarm indicating a broken shear bolt condition. Further in this regard, it should be noted that it is contemplated that controller 40 can include one or more timers or clocks for timing operation of driver 34, and movement and/or non-movement of concave 16, and that memory 42 can include a variety of registers for holding information representative of the various times and positions of concave 16. As examples, such timers can include an initialize shear bolt variables timer; an update previous concave position timer; and a concave not moving timer. Such registers in memory 42 can include, for instance, a current concave position register; and a previous concave position register, either or both of which can be written over as desired. A flip-flop or flag register can also be utilized for storing an indication of a broken shear bolt condition. Referring also to FIGS. 3, 4, 5 and 6, steps of a representative method of operation of system 38 for testing for a broken shear bolt and optionally verifying a broken shear bolt using the above referenced times and registers are set forth. In this example, driver 34 is represented by an electric motor. In FIG. 3, at block 50, the test is initiated. At system startup, it is desirable for the initialize shear bolt variables timer to be set to one second. The process for this is initialized at decision block 52 which determines whether the initialize shear bolt variables timer is not equal to zero, and a key voltage is less than a predetermined value, here, 9.0 volts. If both of these conditions are present, controller 40 will proceed to set the initialized shear bolt variables timer equal to one second, as denoted at decision block 54 and block 56. After the initialize shear bolt variables timer has been set equal to one second, or the key voltage is not less than 9.0 volts, controller 40 will proceed to decrement the initialize shear bolt variables timer by a value of one (equal to 10 milliseconds), as denoted by block 58. Then, the controller will set the previous concave position register equal to the current concave position; set the concave not moving timer equal to 5 seconds as denoted at block 62 ; and set the update previous concave position timer equal to one second as denoted at block 64. Controller 40 will then proceed as denoted at A to end test block 66, then return to block 50 and follow this same sequence of steps as long as the initialize shear bolt variables timer is not equal to zero and/or the key voltage is less than 9 volts. Controller 40 can cycle through this series of steps, including steps 54, 56 and 58, wherein the initialize shear bolt variables timer will be decremented and reset, as long as the key voltage is less than 9 volts. If the key voltage rises to 9 volts or greater, at block 54, controller 40 will bypass block 56 and proceed to decrement the initialize shear bolt variables timer, reset the previous concave position to the current concave position, set the concave not moving timer to 5 seconds, and set the update previous concave position timer equal to one second, as set forth in blocks 58, 60, 62 and 64, then cycle through blocks 66 and 50 and 52, until the initialize shear bolt variables timer has been decremented to zero and the key voltage has remained at 9 volts or above. At block 52, once the initialize shear bolt variables timer is equal to zero, and the key voltage is still 9 volts or above, controller 40 will proceed from block 52, as denoted at C, to decision block 68 in FIG. 4, wherein controller 40 will determine whether a concave shear bolt broken flag is equal to one, denoting a broken condition. If, at block 68, it is determined that the flag is not equal to one, controller 40 will proceed as denoted at D, to block 70 in FIG. 5 wherein it will set the concave shear bolt failure alarm equal to an alarm off condition. Controller 40 will then proceed to decision block 72 to determine whether the previous concave position is less than the current concave position. Here, it should be noted that a lesser position value will denote a concave position closer to the rotor, whereas a greater concave position will denote a position farther from the rotor. If, for instance, referring to FIGS. 1 and 2, driver 34 has not been operated to move the concave, and controller 40 has most recently executed sequence of steps 58 - 64, the previous concave position will have been set equal to the current concave position. As a result, at step 72, the previous concave position should still equal the current concave position. If, on the other hand, driver 34 has been actuated for moving the concave up or down, the previous and current positions should differ accordingly. Also, at the run speed of controller 40, and the operating speed of driver 34 for moving the concave, any change in concave position equal to or greater than 5 millimeters between sequential executions of step 72, can be presumed to be indicative of an abrupt shear bolt failure and resultant fall of the concave. Thus, at decision block 72, if the previous concave position is less than the current concave position, controller 40 will proceed to decision block 74 and determine whether the current concave position minus the previous concave position is greater than or equal to 5 millimeters. If not, any difference will be considered normal and controller 40 will proceed on to the next step. However, if there has been a large change in concave position, controller 40 will proceed to set the concave shear bolt broken flag equal to one which is representative of a broken shear bolt condition, as denoted at block 76. Here, it should be noted that the 5 millimeter value is intended to be a representative value only, and is not intended to limit the present invention. Controller 40 will then proceed to calculate a concave motor current, as denoted at block 78, in preparation for testing whether the concave moves when driver 34 is operated to raise the concave. At decision block 80, controller 40 determines the presence of necessary conditions for this test, including whether the concave motor (driver 34 ) is energized to raise the concave, a concave bridge output is not an error, concave motor current is less than 5 amps, and the current concave position is equal to the previous concave position. If these conditions are not present, controller 40 will set the concave not moving timer to an initial value, here, 5 seconds, as denoted at block 82. Subsequently, controller 40 will determine whether the concave not moving timer is equal to zero, at decision block 84. If controller 40 has proceeded through steps 80 and 82, the concave not moving timer will be set at 5 seconds, such that at decision block 84, it will be determined that the concave not moving timer is not equal to zero, and controller 40 will proceed to the steps contained in FIG. 6. On the other hand, if all of the conditions of decision step 80 are present, controller 40 will decrement the concave position not changing timer by one (10 milliseconds), as denoted at block 86. Then, controller 40 will proceed to determine whether the concave not moving timer is equal to zero, and if yes, will proceed to set the concave shear bolt broken flag to one, representing a broken shear bolt condition, as denoted at block 88 then proceed as denoted at B to execute the steps of FIG. 6. Here, essentially, if the current concave position equals the previous concave position for 5 seconds of operation of driver 34 for raising the concave, controller 40 is determining that a broken shear bolt condition exists. This is a useful test to be conducted when the concave may have been at its lowest position at the time of shear bolt breakage. Referring also to FIG. 6, after execution of the step of block 84 or the step of block 88, controller 40 will proceed to decision block 72 to determine whether the update previous concave position timer is equal to zero, at decision block 93. If no, it will proceed to decrement the update previous concave position timer by one (10 milliseconds), as denoted at block 92 and proceed to the end of the test. If the update previous concave position timer is equal to zero, the controller will set the previous concave position equal to the current concave position, as denoted at block 94. Then, at block 96, the update previous concave position timer will be set equal to one second and the test will end, as denoted at block 66. Referring again to FIGS. 3 and 4, after initiating the test, as denoted at block 50 ; determining that both conditions of decision block 52 are not present; and having a concave shear bolt broken flag condition equals one (indicating a broken shear bolt condition) controller 40 will proceed through a sequence of steps to determine whether the broken shear bolt flag setting is erroneous. Here, at decision block 98, controller 40 determines whether the thresher is engaged. If yes, a priority 3 concave shear bolt failure alarm is outputted, as denoted at block 100. This is a high level alarm condition. If, on the other hand, at block 98 it is determined that the thresher is not engaged and running, a priority 1 concave shear bolt failure alarm will be outputted, as denoted at block 102. Then, controller 40 will determine whether the concave motor is energized, at decision block 104. If not, it will proceed, as denoted at B, to the sequence of steps shown in the diagram of FIG. 6. If, at block 104, it is determined that the concave motor is energized, controller 40 will proceed to determine whether the current concave position is not equal to the previous concave position, at decision block 106. If it is determined that the current and previous concave positions are equal, it will be determined that the broken shear bolt condition is true, and controller 40 will proceed to execute the steps of FIG. 6. If, at block 106, it is determined that the current concave position is not equal to the previous concave position, this is an indication that the concave has moved responsive to operation of driver 34. Accordingly, controller 40 will proceed to set the concave shear bolt flag to a zero condition, indicating that the shear bolt is intact, as denoted at block 108. The previous concave position will then be set equal to the current concave position, as denoted at block 110, and controller 40 will proceed to execute the steps of FIG. 6. From the proceeding discussion, it should be apparent that control system 38 is operable according to the steps of the present invention, to diagnose a shear bolt failure or breakage condition as a result of an abrupt or rapid downward movement of the concave, as denoted by the sequence of steps 70, 72, 74 and 76. Additionally, if the concave is at or adjacent to the bottom of the range of normal positions thereof, a broken shear bolt condition can be diagnosed by the steps 80, 82, 84 and 86. Still further, if a broken shear bolt condition flag exists, the existence of a broken shear bolt, or non-existence thereof, can be determined by the steps of FIG. 4. As a result of the operating steps of the system according to the present invention, a shear bolt failure condition can be accurately and quickly diagnosed and determined using the components used for moving and determining the position of the concave. It will be understood that changes in the details, materials, steps, and arrangements of parts which have been described and illustrated to explain the nature of the invention will occur to and may be made by those skilled in the art upon a reading of this disclosure within the principles and scope of the invention. The foregoing description illustrates the preferred embodiment of the invention; however, concepts, as based upon the description, may be employed in other embodiments without departing from the scope of the invention. Accordingly, the following claims are intended to protect the invention broadly as well as in the specific form shown. | Summary: A system and method for detecting existence of a failed or broken shear bolt supporting all or a portion of a concave of a threshing system of an agricultural combine and utilizing information relating to rates of movement and absence of movement of a concave to determining the existence and non-existence of a failed or broken shear bolt supporting the concave. More particularly, the present system and method is operable to diagnose existence of a broken shear bolt from a rapid downward movement of the concave and non-movement of the concave during operation of a driver for repositioning the concave. Also, existence of a false broken concave condition can be diagnosed by movements of the concave responsive to operation of the driver. | 6,127 | 152 | big_patent | en |
Write a title and summarize: Cell surface protein and lipid molecules are organized in various patterns: randomly, along gradients, or clustered when segregated into discrete micro- and nano-domains. Their distribution is tightly coupled to events such as polarization, endocytosis, and intracellular signaling, but challenging to quantify using traditional techniques. Here we present a novel approach to quantify the distribution of plasma membrane proteins and lipids. This approach describes spatial patterns in degrees of inhomogeneity and incorporates an intensity-based correction to analyze images with a wide range of resolutions; we have termed it Quantitative Analysis of the Spatial distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). We tested its applicability using simulated microscopy images and images acquired by widefield microscopy, total internal reflection microscopy, structured illumination microscopy, and photoactivated localization microscopy. We validated QuASIMoDOH, successfully quantifying the distribution of protein and lipid molecules detected with several labeling techniques, in different cell model systems. We also used this method to characterize the reorganization of cell surface lipids in response to disrupted endosomal trafficking and to detect dynamic changes in the global and local organization of epidermal growth factor receptors across the cell surface. Our findings demonstrate that QuASIMoDOH can be used to assess protein and lipid patterns, quantifying distribution changes and spatial reorganization at the cell surface. An ImageJ/Fiji plugin of this analysis tool is provided. The function of cell surface proteins and lipids is tightly coupled to their spatial organization [1–3]. Membrane constituents cluster in nano- and micro-domains originating from lipid affinity (e. g., lipid rafts) [4], protein-protein interactions (e. g., tetraspanin domains) [5], and constraints imposed by the cytoskeleton [6]. Plasma membrane organization is also inherently asymmetric in polarized cells, such as migrating cells [7,8], epithelial cells [9], and neurons [10,11]. The ability to detect this plasma membrane organization is crucial for unraveling the dependency of signaling events, and understanding membrane regulation in a disease-related context [12]. One approach to assess the distribution of plasma membrane molecules is to consider these as bi-dimensional point processes that can be analyzed by spatial statistics. Point processes can be classified as: (I) Homogeneous or random, characterized by a constant density of points; (II) Inhomogeneous, characterized by a non-constant density of points; (III) Regular, with points equally dispersed; and (IV) Clustered, where points are grouped [13]. For investigating the spatial organization and especially for studying clusters, Ripley’s K-function [14] and pair correlation (PC) function approaches have been established. These measure the number from neighbors within a certain distance of a protein to determine the amount of clustering [15,16]. Several modifications and extensions of Ripley’s K-function have been made, including a model-based Bayesian approach [17], the extension to co-clustering [18], and an adaptation to account for limited localization precision in single molecule localization microscopy [19]. The PC function has been applied to images acquired by Photoactivatable Localization Microscopy (PALM) to quantify the heterogeneity of protein distributions on the plasma membrane [20]. In addition, the Density-Based Spatial Clustering of Applications with Noise (DBSCAN) algorithm, a density based tool, was used to identify clusters of varying shape against a background in super-resolution microscopy [21]. Recently, the Ordering Points To Identify the Clustering Structure (OPTICS) algorithm was made available to the single molecule community to measure local density changes (overcoming some of the limitations of DBSCAN) [22,23]. Moreover, the Getis-Ord G statistic [24] has been used to quantify the degree of local protein clustering in super-resolution images [25] and compared to other methods including DBSCAN and PC analyses. A key limitation of the current toolset is the lack of assessment on both clustering and polarity, equally important from a biological point of view. While the ability to detect specific clusters is of great importance, Sengupta et al. [20] among others, have shown that molecules like glycosylphosphatidylinositol (GPI) -anchored proteins are organized into different populations of clusters as well as residing as single molecules. All these organizations should be accounted for when analyzing the overall distribution. Furthermore, current tools require knowledge of the precise localization of the proteins and are thus limited to super-resolution or electron microscopy images. Therefore, we aimed to create an analysis tool that investigates biomolecule distributions without upfront information about their organization, and that can be applied to a variety of microscopy methods, including super-resolution and widefield microscopy. The main principle of our approach is to investigate spatial patterns of proteins and lipids and to quantify any deviation from random towards clustered or polarized organization as a measure of increased inhomogeneity. We exploit a geometrical approach, called tessellation, to divide the image into tiles, and use the distribution of tile areas to characterize different patterns. The image analysis algorithm uses both the information of the neighbor relations of segmented objects and the intensity of the tiles in which they are confined. By using fluorescence intensity information for tile area correction, we have made the tool applicable to images acquired by a range of microscopy techniques. We have termed this analysis tool Quantitative Analysis of the Spatial-distributions in Images using Mosaic segmentation and Dual parameter Optimization in Histograms (QuASIMoDOH). An ImageJ/Fiji [26] plugin for QuASIMoDOH analysis is available along with instructional documentation (S1 File). For QuASIMoDOH analysis we consider single fluorescent emitters distributed on the cell surface as a point process P in a bi-dimensional space. Considering a number N of individual points p of the process placed on the support S, the distribution pattern P is defined as: P=∑i=1Npi with pi ∈ S, ∀i∈{1,2, 3…N}, (1) where P describes a homogenous, clustered, or inhomogeneous pattern. Considering the brightness f of the fluorescent emitters (represented by p) and assuming the emitters being equally bright, the distribution pattern can be defined as: P=∑i=1Npif with pi ∈ S, ∀ i ∈{1,2, 3…N}. (2) When the point process P (S1A Fig) is imaged by an optical system, each point of position r, p (r), will be diffracted by the Point Spread Function (PSF). The resulting microscope image g (r) is typically approximated by the convolution (⊗) of the point p (r) with the PSF [27]: g (r) =p (r) ⊗ PSF (r). (3) The result is a blurred image (S1B Fig). Due to the band limitation of the PSF, points of the pattern P placed at a distance shorter than the band limit will not be resolved as single points. In the blurred image, the intensity of the pixels depicting the diffraction pattern is directly related to the number of points contributing to this diffracted pattern. Aiding the development of QuASIMoDOH, we generated in silico images of points (shown as single pixels) dispersed on a surface (Fig 1A, S1 Text). Blur and noise were then added into the image (Fig 1B) to mimic the acquisition process of a fluorescence microscope (see S1 Fig and S1 Text for detailed description). The individual steps of QuASIMoDOH analysis are depicted in Fig 1 and described in detail below: To verify the ability of QuASIMoDOH to detect different distributions, we generated in silico images of different point patterns (see ‘Creation of discrete point pattern images’ in S1 Text and S3 Fig for a detailed description of the patterns). As described before, blur and noise were added to simulate the acquisition process of a fluorescence microscope (S1 Text, S1 Fig). We simulated images from widefield (WF) and Total Internal Reflection Fluorescence (TIRF) microscopy (resolution ~200 nm), Structured Illumination Microscopy (SIM) (resolution ~100 nm), and PALM (resolution ~20 nm). We decided to generate biologically relevant patterns, namely: random distributions (Fig 2A); random distributions of clusters with diameter d = 80 nm (Fig 2B); random distributions of clusters with diameter d = 240 nm (Fig 2C); polar distributions (Fig 2D); and polar distributions of clusters with diameter d = 240 nm (Fig 2E) for WF (Fig 2F–2J) and SIM images. For PALM, clusters with increasing sizes were simulated (see S1 and S3 Figs and S1 Text). The correction factor used in the analysis of simulated images was determined by maximizing the accuracy of the analysis to give the correct number of known points. The accuracy is defined as the ratio between the number of areas, obtained by tile size correction, and the total number of points in the image (see S1 Text and S4 Fig). When the tessellation (Fig 2K–2O) and intensity correction are applied to images of different point patterns, the result is a set of tile areas typical for each pattern, captured by the histograms of corrected tile areas (Fig 2P–2T). The histograms display an increasingly steep peak for increasingly inhomogeneous patterns, moving from random to polar clusters. Modeling the distribution of tile areas by the Inverse Gamma PDF allows for the discrimination between different point patterns. The two parameters of the function are specifically associated with the histogram fit shape and its broadness. These parameters ultimately vary based on the underlying point pattern. In particular, they decrease with an increase in the inhomogeneity of the distribution, as shown in Fig 2U where the shape and scale parameters are plotted together. The average (and standard error of the mean, SEM) from the analysis of 50 images for each point pattern is presented in Fig 2U. The analyzed images have the same density, ρ, defined as: ρ=NA/AreaImage, (10) where NA is the number of areas obtained upon tile size correction and AreaImage is the total area of the image in μm2. Fig 2U shows the results from analyzing images with density ρ ~5 (tiles/μm2); the images in Fig 2F–2J are examples of this density. Fig 2V shows a plot of the shape and scale parameters obtained by analyzing images with different patterns and densities (1 ≤ ρ ≤ 7 tiles/μm2). The value of the shape parameter increases with increasing density, while the scale parameter decreases. Examples of images of density ρ ~1 and ρ ~7 (tiles/μm2) are given in Fig 2W top and bottom, respectively. S5 Fig shows the densities associated with each point on the reference graphs for microscopy techniques with different resolution. The deviation from a random distribution (calculated as the Euclidean distance from the random reference point) is used as a measure of the inhomogeneity of a point pattern. This is quantified in Fig 2X. Here the inhomogeneity is calculated for images with ρ ~5 (tiles/μm2) (Fig 2U). To provide comparable results for each density, the interval between the two extremes (random and polar clusters) was normalized to one. Analysis of images similar to the one shown in S6A Fig can result in shape and scale parameters larger than shape and scale parameters for the corresponding random distribution reference (S6B Fig). The inhomogeneity measure, in this case, will have a negative value (S6C Fig) since the coordinates of the random distribution are used as the origin from which the inhomogeneity is calculated. Shape and scale parameters 6–8 times larger than the random distribution reference can be further explained by a regular pattern where points are equally dispersed (S6D–S6F Fig). Taken together, these results show that QuASIMoDOH can be used to quantify the inhomogeneity of spatial distributions and demonstrate the validity of the approach at the level of simulated images. We subsequently validated our method using images from fluorescence microscopy. Real and simulated images can exhibit analogous features, but they may differ as a consequence of microscope/detector sensitivity and dynamic range. Therefore, the correction factor C for actual microscopy images was selected to establish a comparable degree of correction as that determined by simulated results (see S1 Text and S7 Fig). We took advantage of plasma membrane sheets (S8 Fig) [36,37] to acquire images of the cell surface without fluorescent background arising from the cytoplasm [38,39]. To control for the integrity of the plasma membrane, cells were initially incubated with the lipophilic fluorescent stains DiO or DiI, depending on the combination of subsequent protein/lipid staining. We tested whether QuASIMoDOH analysis was able to replicate findings made using microscopy techniques with different resolution and measured the inhomogeneity value from similar samples. We computed the cluster size of the lipid raft marker glycosylphosphatidylinositol (GPI) -anchored protein by comparing the results against the reference graph for simulated WF, SIM, and PALM images (S5 Fig). Images of GPI-anchored protein tagged with green fluorescent protein (GFP) or photoactivatable GFP (paGFP) [40], were acquired by WF, SIM, and PALM (S9A–S9C Fig). QuASIMoDOH analysis of SIM and WF data gave comparable results (S9D and S9E Fig). The analysis of 16 PALM images resulted in a GPI cluster size between 50 and 100 nm in diameter (two regions were identified as random and one region with a low r2 was excluded) (S9F Fig). PC-PALM analysis of the same 16 images resulted in an average GPI cluster size of approximately 80 nm in diameter (one region was identified as random). Obtained values for GPI cluster size were also consistent with previously published work [41,42]. Thus, QuASIMoDOH is capable of providing fast and quantitative analysis of super-resolution data with respect to cluster size. Finally, GPI inhomogeneity measurements from WF, SIM, and PALM data provided similar results when using appropriate tile area intensity corrections (see S9G Fig), indicating the applicability of the approach across microscopy techniques with various resolutions. To further confirm that QuASIMoDOH detects different distributions we labeled three proteins that have well described and distinct spatial arrangements in Mouse Embryonic Fibroblasts (MEFs) using indirect immunostaining, and imaged these by WF microscopy. We used the sodium-potassium pump (Na+/K+ ATPase) [43] (Fig 3A and 3B), the transferrin receptor (TfR) [44] (Fig 3C and 3D), and caveolin1 (Cav1) [45] (Fig 3E and 3F). The Na+/K+ ATPase is an integral membrane protein that is randomly distributed across the cell surface. The TfR is an integral membrane protein responsible for ferric ion uptake, which clusters in clathrin-coated pits (200 nm in size) after ligand binding and prior to endocytosis. Cav-1 is a cytosolic peripheral-membrane protein that functions, together with cavin, in the formation of caveolae. Cav-1 is predominantly localized to the trailing edge of migrating cells, and thus shows a polarized distribution. Analysis by QuASIMoDOH demonstrates that the different distribution patterns of immunolocalized Na+/K+ ATPase, TfR, and Cav-1 can be reliably distinguished based on their inhomogeneity (Fig 3G and 3H). Comparing the results to simulated images indicates that the distribution pattern of Na+/K+ ATPase most closely resembles simulated images with a random distribution, whereas TfR matches those with a clustered distribution, and Cav1 aligns with simulated images highlighting a polar distribution of clusters. Thus, QuASIMoDOH effectively captures different protein distributions. Furthermore, these results demonstrate that our analysis can be successfully applied to images of proteins detected by immunolabeling. The cell surface is also patterned by the wide variety of lipid molecules present in the plasma membrane. We, therefore, explored whether QuASIMoDOH detects lipid distributions, including changes that result from perturbation of lipid trafficking (Fig 4A–4D). For this purpose, we treated MEF cells with U18666A, an amphipathic steroid that causes cholesterol and sphingomyelin accumulation in late endosomes and lysosomes [46,47] (3 μg/mL for 18 h). The treatment was followed by fixation and incubation with Lysenin (a toxin that specifically binds sphingomyelin in the membrane [48,49]) and immunostaining. We reasoned that blocking endosomal sphingomyelin trafficking would deplete this lipid from the cell surface where it is normally organized into clusters, possibly causing reorganization to a more homogeneous distribution. Indeed, QuASIMoDOH analysis detects a significant difference in sphingomyelin organization between drug-treated and control cells (t-test: p = 0. 045; two-tailed; unpaired, Fig 4E and 4F). Thus, QuASIMoDOH analysis can also detect plasma membrane lipid distributions and how these are altered in response to cellular perturbations. To further explore QuASIMoDOH analysis in combination with different techniques we turned to TIRF imaging (with widefield resolution). We monitored the internalization of the epidermal growth factor receptor (EGFR) [50] (S10 Fig). We performed this in HeLa cells transiently transfected with EGFR-GFP, and treated with two concentrations of EGF reported to direct EGFR internalization distinctly [51,52]: a low EGF dose (2 ng/ml) induces EGFR internalization via the clathrin-mediated route, while a high EGF dose (20 ng/ml) directs EGFR internalization through both clathrin-coated pits and caveolae. TIRF images of cells fixed at specific time points (t0, before stimulation, t1 = 2, t2 = 5, t3 = 7, t4 = 10, and t5 = 15 minutes following EGF addition) were acquired, and the organization of EGFR at the plasma membrane was analyzed by QuASIMoDOH (Fig 5). This revealed that EGF indeed altered the EGFR distribution (Fig 5G–5J), corresponding to an increase in the measure of inhomogeneity. Furthermore, as predicted, this occurred more rapidly for cells exposed to the high concentration of EGF. These results demonstrate that QuASIMoDOH can be used to follow dynamic processes occurring at the cell surface and distinguish those processes with different rates. The EGFR internalization assay raises the question: Can different spatial distributions observed in the plasma membrane (Fig 5E and 5F) be analyzed on a local scale as opposed to the global scale described above? To address this, we expanded QuASIMoDOH analysis to assess local distribution differences. For a local analysis, the tile area distribution analysis (see above ‘5. Tile area distribution analysis’) is applied to a subset of tiles in the images. This subset of tiles is selected by a circle that moves from the center of one tile to the next (Fig 6A and 6B). A color is assigned to the tile in the center based on the distribution detected using the selected tiles. Magenta represents a random distribution, green represents a distribution in clusters (for simplicity, the distinction between clusters of different sizes is omitted), and red represents polar distributions (for simplicity, the distinction between polar and polar clusters is omitted). When switching to a local analysis, the fitting quality (see S11 Fig) can be affected. To limit incorrect distribution assignment, we set 0. 45 as a minimum r2 and a cut off for the outliers that, despite a high enough r², are too far away from any reference point (distance > 1, for widefield images), resulting in tiles without color. To test the local analysis, we first created in silico images where the upper part contained a random distribution and the lower part consisted of a clustered distribution (see example in Fig 6A). These test images were simulations of WF images with a size of 512 x 512 pixels and 1600 points, on average. To maximize the number of tiles with detected distributions, the diameter of the circle must be in the range of 4 to 10 μm (Fig 6C, S1 Text and S12 Fig). As expected, from the local analysis we obtained a random distribution in the upper third of the image, a clustered distribution in the lowest third, and a polarized distribution in the center due to the neighborhood abrupt change from one part of the image to the other in this transition region (Fig 6D). We next applied the local analysis to a fixed cell image from our EGFR internalization assay (treated for 10 min with high dose of EGF, Fig 6E). In Fig 6F, we applied the local analysis by setting the circle diameter in the range of 4 to 15 μm (similar to the simulation, the maximum diameter is about half of the image). A clear clustered organization of the receptor becomes apparent at the periphery of the cell following the analysis. A random distribution, however, covers the center of the cell and a gradient of random and clustered receptors mark the transition between these regions. Finally, an image of EGFR on the surface of a live cell, stimulated by 20 ng/mL of EGF, was used to further investigate the successful application of this local analysis approach. We were able to observe the local variation of receptor distributions following the time dependence of stimulation (see S1 Movie). These results indicate that QuASIMoDOH can be used to assess both the global and local changes in the distribution of fluorescent patterns at the plasma membrane. Here we present QuASIMoDOH as a new approach to measure the inhomogeneity of a spatial distribution as a deviation from random towards clustered and polarized patterns (S13 Fig). Different from methods such as PC-PALM [20], Ripley’s K-function [14], nearest neighbor approaches [13], and DBSCAN [22,23], QuASIMoDOH is compatible with polarized distributions (see Caveolin-1 in Fig 3). It can detect and measure polarized distributions independent from their orientation, while other tools must acquire information on cell morphology or other features to assess the spatial phenotype of polarized molecules [53]. Compared to grid-based algorithms, like the Hoshen-Kopelman algorithm [54] that divides space into a grid and identifies clusters as continuously occupied areas, QuASIMoDOH can correct for unresolved points by complementing the tile area dataset with the information on tile intensity and is thus applicable beyond single molecule imaging techniques. Where SpIDA [55] measures protein interactions and aggregation by multiple fitting of pixel intensity histograms, QuASIMoDOH analyzes the fit from tile intensity histograms to extract a measure of plasma membrane inhomogeneity. Comparable to Number and Brightness (N&B) approaches [31], which analyze temporal fluctuations, in QuASIMoDOH the tile intensity IT depends on the number of molecules (p) in the tile and their brightness (f): IT = pf. After calibration, incorporating background intensity, N&B can be mapped to absolute values with good spatial resolution. For ease of use in QuASIMoDOH, we decided to estimate the correction factor from the image by an analysis of the distribution of tile intensities. Determining the correction factor, however, leaves the possibility of misinterpretation due to background, non-uniform illumination, or the presence of artifacts. Pre-processing steps to address these conditions are provided in the QuASIMoDOH documentation (S1 File). S14 Fig offers information on how to determine if an image is suitable for QuASIMoDOH analysis. We applied QuASIMoDOH to PALM, SIM, WF, and TIRF images (Figs 3–5, S9 Fig). However, in principle, this tool can also be applied to other microscopy modalities, including confocal microscopy and (d) STORM imaging. We have demonstrated that results obtained for the cluster analysis of GPI-anchored proteins are in reasonable agreement across microscopy techniques with different resolution and with previously published results obtained by PC-PALM analysis [42]. Additionally, QuASIMoDOH correctly detected the differential distribution of Na+/K+ ATPase, TfR, and Cav-1 proteins. Notably, QuASIMoDOH does not require the presence of a reference molecule in the sample with a known distribution. As a result, comparisons between protein/lipid organization in different cell types or within the same cell and under different experimental conditions are possible, e. g., evaluating changes in a lipid distribution following drug treatment. We specifically demonstrated this by analyzing the distribution of sphingomyelin (Fig 4). The observed increase in homogeneity for the lipid probe Lysenin in cells treated with U18666A compared to untreated cells emphasizes the potential of our analysis tool to readily test a range of cell perturbations. Additionally, the fact that affinity probes, fusion proteins, and lipid probes can be detected highlights the versatility of our analysis method. We further demonstrated the ability of this approach to reveal the dynamic nature of EGFR organization at the plasma membrane upon stimulation, using both fixed and live cells. A list of advantages and limitations of QuASIMoDOH is provided in Table 1. An important feature of QuASIMoDOH analysis is its direct application to microscopy data with no detailed prior knowledge of the sample. Overall QuASIMoDOH serves as a quick, straightforward, and automatable method to measure distribution patterns of proteins and lipids on the cell surface. It can be used to study events at the cell surface related to cell signaling or remodeling as these appear, for example, in the reprogramming of cancer cells or neuronal differentiation. For QuASIMoDOH development and testing, a Dell Optiplex7010 computer was used (Intel CITM) i7-3770 CPU @ 3. 40GHz processor and 4. 00 GB RAM, running Windows 7 Professional). Images were run in batch mode, each 256 x 256 pixels in size. In total, approximately 25,000 points were identified, and using a standard four year old personal computer, we were able to analyze the entire dataset of 50 images in 22 seconds. Pixel-by-pixel image processing of both simulated and fluorescent images was carried out using custom-made routines in MATLAB and ImageJ/Fiji [26] according to the schemes described in the main text. For plasma sheet samples, background correction was applied (where necessary) by subtracting a background value either using the Dip Image function “backgroundoffset” or manually. Background subtraction on TIRF images of intact cells was carried out using the ImageJ/Fiji function “Rolling Ball” [26]. The WF, SIM, and TIRF images are initially filtered by the ImageJ/Fiji filter ‘Sigma Filter Plus’ and then smoothed. For the separation of signal and background in our simulated images, we used the default threshold in ImageJ/Fiji [57], which is based on Isodata. For the widefield images of Na+/K+ ATPase and Caveolin-1, the threshold Li [58] was used. GPI, TfR, and Lysenin widefield images, as well as GPI SIM and EGFR TIRF images, were thresholded using Mean [59]. Apparently, the best-suited threshold for an image can depend on the imaging modality and was chosen upfront based on the obtained images (see S1 Text). For skeletonization, the function implemented in ImageJ/Fiji version 1. 47 was used. PALM super-resolution images were generated by analyzing datasets obtained from Peak Selector software (Research Systems, Inc.). Analysis of paGFP-GPI images was performed on 16 square regions of 7–18 μm2 obtained from 8 cells, with an average of 75 ± 5 localized peaks/μm2 and average localization precision of 15 nm. PC-PALM analysis was performed similarly to the previously described method [20]. For QuASIMoDOH analysis, localized peaks were grouped using a group radius of 3 x maximum localization precision and a maximum dark time of 5 s using Peak Selector. The maximum dark time was obtained experimentally using sparse paGFP. Similar to the method previously reported by Annibale et al. [60], a best fit of the observed fluorophore counts as a function of the dark time was used to determine the effective number of molecules present in the sample. Grouped peak coordinates (in pixels) were subsequently fed into ImageJ/Fiji for QuASIMoDOH analysis. Peaks were plotted following a conversion to nanometers by a user-defined pixel size. The image width and height was calculated from the coordinates as the difference between the maximum and minimum values of X and Y coordinates, respectively. The generated image was then scaled to a 2. 5 nm pixel size, and subsequently analyzed as described before. All graphing and statistics were prepared using MATLAB, GraphPad Prism (GraphPad, La Jolla, USA), and Excel (Microsoft, Redmond, USA). For the analysis of distribution patterns of proteins and lipids, images of cells from three different preparations/coverslips were analyzed. Images were pooled for further QuASIMoDOH analysis. Mouse Embryonic Fibroblast (MEF) cells were maintained at 37°C and 5% CO2 in DMEM/F12 (+L-glutamine + 15 mM HEPES) supplemented with 10% of fetal bovine serum (FBS). MDA-MB-468 cells were cultured in DMEM supplemented with 10% FBS. Transient transfection of MDA-MB-468 cells was performed using Jetprime (PolyPlus, following manufacturer instructions) with 2 μg of paGFP-GPI similarly as described in detail elsewhere [42]. Transient transfection of MDA-MB-468 cells was performed using FuGENE (Promega, following manufacturer instructions) with 2 μg of GFP-GPI. HeLa cells were grown in DMEM/F12 (+L-glutamine + 15 mM HEPES) supplemented with 10% FBS. One day after plating cells, transfection was performed using FuGENE6 with 0. 5 μM of plasmid pGFP encoding for EGFR-GFP. Plasma membrane sheets were prepared as previously described [36]. In short, MEF cells were cultured in DMEM/F12 (+L-glutamine + 15 mM HEPES) supplemented with 10% of fetal bovine serum. Cells were grown on coverslips to approximately 60% confluence. At 4°C, cells were washed with PBS+/+ and subsequently with coating buffer (20 mM MES, 135 mM NaCl, 0. 5 mM CaCl2,1 mM MgCl2, pH5. 5). Next, they were incubated in coating buffer with 1% of silica beads for 30 min. They were rinsed with deionized water (10 min) followed by three washing steps with PBS+/+. To prepare plasma membrane sheets, shear force was applied to the coverslip using a syringe held at a 30° angle (on the coverslip). As the upper surface of adherent cells is made rigid by the silica coating, shear forces break off membranes at the edges releasing all soluble contents and retaining only the basal plasma membrane adherent to the coverslip. These remaining sheets were then fixed (4% paraformaldehyde in PBS, 15–20 min at RT) (see schematic in S8 Fig). Fixed plasma membrane sheets were rinsed with PBS+/+ and blocked (2% FCS, 2% BSA, 0. 2% gelatin, 5% goat serum in PBS-/-) for 1 h. For immunofluorescence, the following antibodies were used: mab to murine Cav-1 (BD Biosciences; San Jose, USA) (1: 200); mab against Transferrin receptor (Zymed/Life Technologies) (1: 100); mab against Na+/K+ ATPase alpha (Novus Biologicals, Littleton USA) (1: 100). Alexa488/555/568 conjugated secondary antibodies were used (Life Technologies) (1: 1000). Lysenin (1: 40) was purchased from Peptide Institute (Osaka, Japan) and immunolocalized using anti-Lysenin pab (1: 100) followed by goat anti-rabbit Alexa-555 (Life Technologies) secondary antibody. U18666A was purchased from Sigma. DiI and DiO (Life Technologies) (1: 100) were used to stain lipid bilayers. Staining was performed according to manufacturer recommendations. Twenty-four hours after transfection, HeLa cells were serum starved overnight. For the preparation of the fixed samples, after starvation, the cells were incubated at 37°C with either 2 ng/mL or 20 ng/mL EGF for different time intervals (2,5, 7,10, and 15 minutes), then fixed with 4% paraformaldehyde in PBS, for 15–20 minutes at room temperature. The fixed samples were then imaged by TIRF. Additionally, living cells were imaged by TIRF upon stimulation with 20 ng/mL of EGF (S1 Movie). Widefield and structured illumination images were acquired with a structured illumination microscope (Elyra S1 (Carl Zeiss, Jena, Germany) equipped with a 63x oil objective lens with a numerical aperture (NA) of 1. 4 and an Andor iXon 885 EM-CCD camera). PALM imaging was performed using a Nikon Instruments Ti Eclipse inverted microscope with a 100x/1. 49 NA TIRF objective (Apo) and a 488 nm laser (Agilent, MLC-MBP-ND laser launch) with an EM-CCD camera (Andor Technology, iXon DU897-Ultra). The microscope was equipped with a Perfect Focus Motor to minimize axial drift over the duration of imaging. paGFP was simultaneously activated and excited with the 488 nm laser at an intensity set to 1. 45–1. 9 mW (as measured at the optical fiber). Exposure time was set at 100 ms. Imaging was performed until paGFP was completely exhausted, typically after 20,000 frames. TetraSpeck beads (Life Technologies) were used as fiducial markers for drift-correction during image acquisition. TIRF images were acquired by a Nikon Ti Eclipse inverted microscope, equipped with a 100x/1. 49 NA oil objective lens and a Hamamatsu Orca D2 camera, or Hamamatsu Orca Flash 4 Lite, for living cell imaging. Confocal images were acquired using the same microscope equipped with Nikon A1R using a 60x 1. 4 NA oil objective. Co-localization analysis was carried out using the Fiji plugin JACoP [61]. | Title: Inhomogeneity Based Characterization of Distribution Patterns on the Plasma Membrane Summary: Plasma membrane organization is fundamental to cellular signaling, transport of molecules, and cell adhesion. To achieve this, plasma membrane proteins and lipids are spatially organized: they form clusters, aggregate in signaling platforms, distribute into gradients on polarized cells, or randomly distribute across the membrane. It is also clear that these organizations can be affected in various contexts. For example, in aging or neurodegenerative diseases, the composition of the plasma membrane is altered and, consequently, the protein and lipid distributions in the membrane fluctuate. In addition, cancer progression is characterized by changes in cellular polarity, lipid content, and the redistribution of cell surface receptors and adhesion molecules. Here we have developed a method to quantify such alterations that, unlike current tools, is compatible with diverse types of cellular organization, including polarity. Our tool can be employed to screen for changes in a straightforward manner and to elucidate distributions of cell surface components in different disciplines, ranging from neurobiology to cancer research. | 8,241 | 237 | lay_plos | en |
Summarize: BACKGROUND OF THE INVENTION Disposable diapers have met with increasing commercial acceptance in recent years, primarily because of their convenience. Such diapers have conventionally included a facing layer to be brought into contact with an infant's skin, an absorbent panel adjacent thereto, and a water-impervious or a water repellent outer layer. Known types of disposable diapers have had many functional deficiencies including inadequate absorptive capacity and inability to keep moisture away from the surface of the diaper which comes into contact with the infant's skin. Another serious drawback of prior art diapers is the tendency for liquid to leak around the edges of the diaper, particularly at night during periods of heavy discharge. A significant advance in the art is provided by the diaper constructions disclosed and claimed in commonly assigned, Mesek et al., U.S. Pat. No. 3,612,055. The diaper structure illustrated therein includes, in order; a fibrous facing layer which is to be brought into contact with the infant's skin; an absorbent panel comprising a batt of highly porous, loosely compacted cellulose fibers having a paper-like, densified, highly compacted cellulosic fibrous layer integral with the loosely compacted batt; and an impervious backing sheet adhered to the densified layer portion of the batt throughout the interface therebetween. The facing layer is of porous construction and its fibers have less wettability for water than the fibers of the loosely compacted batt, resulting in a tendency for liquid to flow from the facing web into the batt. The densified fibrous layer has a smaller average pore size than the loosely compacted batt resulting in a tendency for liquid to flow from the batt into the densified layer. In one embodiment of the diaper disclosed in the above mentioned patent, having particular utility during periods of heavy discharge, the absorbent panel of the diaper includes a relatively small second batt, similar to the batt already named, superimposed on the larger first named batt. This construction not only provides an increased absorptive capacity for the diaper, but also provides for greater compressibility at the center of the diaper because of the increased batt thickness. When the batt portion of the diaper is compressed by the infant's weight, the distances between adjacent fibers is decreased, i.e., there is a smaller effective capillary radius between adjacent fibers, particularly in the center section of the batt portion of the diaper. In consequence of this, there is a greater wickability at the more highly compressed center portion of the batt as compared to the less compressed marginal portions. This latter construction tends to keep liquid in place in the center portion of the diaper, and prevents it from leaking around the edges thereof. In the last mentioned diaper embodiment, the integral densified layer portion of the larger batt is in face-to-face engagement with the backing sheet, thus helping the urine to spread laterally throughout the length and width of the batt beyond the edges of the smaller batt. The rapid spread of the urine by means of the densified layer is desirable, but carrying the liquid to the peripheral edges of the larger batt increases the likelihood of leakage at the edges of the diaper. An improvement in multi-layer batt diapers is disclosed in commonly assigned copending, Mesek Application Ser. No. 187,248. The diaper structure utilized therein includes an absorbent panel consisting of two differently sized, superposed batt layers of highly porous, loosely compacted cellulose fibers, sandwiched between a porous facing layer and a water-impervious sheet, with the smaller of the batt layers being positioned adjacent the backing sheet, and with the larger batt layer being positioned over the smaller batt layer. In this last mentioned embodiment, the added thickness provided by the smaller batt effectively confines large volume discharge of urine in areas out of contact with the infant's skin. However, due to the two-piece construction of the batt portions of this diaper, a large amount of cellulose fibers must be used, and this embodiment requires complex production apparatus to provide proper cutting and positioning of the two batts in registry with one another and the other components of the diaper. SUMMARY OF THE INVENTION The diaper of the present invention represents an improvement to the single batt, heavy discharge type of diapers by virtue of minimizing the likelihood of urine leaking from the edges of the diaper. To achieve this important result, the diaper of the present invention includes an absorbent panel consisting of a double contoured batt layer of highly porous, loosely compacted cellulosic fibers, sandwiched between a porous facing layer and a water impervious sheet. In consequence of the construction of the diaper of the present invention, urine passes into the double contoured cross-sectional absorbent batt through the facing layer, flows preferentially into the densified layer of the batt to draw the liquid away from the infant's skin. Urine flowing into the densified layer tends to spread laterally because of its wicking action. The increased compressibility resulting from the double contoured cross-sectioned batt at the central portion of the diaper, combined with the compression caused by the infant's weight, provides for greater wickability at the longitudinal and transverse central portions of the diaper, so that there is a cooperative relationship with the densified layer which tends to concentrate urine away from the side edges of the diaper. Further, the non-contoured extremities of the batt (which have less cellulosic fibers than the contoured portion) provide, in effect, a barrier which also contributes to the retention of urine in the central portions of the diaper. The construction of the diaper of the invention, as a whole, provides a mechanism for rapidly transporting urine from the point of discharge from the infant, and for spreading urine throughout most of the absorbent panel, while at the same time retarding the flow before the urine reaches the edges of the batt. It also provides a mechanism for holding urine discharge of limited content within the median portions of the diaper by a combination of a densified layer and a greater overall batt density in the median regions, provided by the action of the infant's weight on the double contoured cross-sectional portions of the diaper. In addition to the advantages described above with respect to the handling of urine discharge by the diaper of this invention, it also provides enhanced structural stability, as compared to the above mentioned two-layer batt diapers which permit relative movement between the batts at their interface, as well as more efficient and easier production. The batt is directly adhered to the backing sheet (which is ordinarily the strongest structural element of the diaper), at the interface therebetween. Thus, the batt is positively anchored to the backing sheet against movement and against disintegration. The increased structural integrity is of special importance in a diaper that can hold a large volume of urine since the increased weight of the urine-saturated diaper subjects it, and particularly its relatively flimsy absorbent panel, to increased stress. Moreover, since the batt is integrally formed (as opposed to the two-layer batt panels of the prior art), there is no movement between the contoured cross-sectional medians and the marginal portions of the batt. Additionally, since the batt is a single unit, the amount of fibers which are used in a batt at a given maximum cross section is reduced, as compared to a two-layer batt construction, with a resulting decrease in production cost, and registration problems, inherent in the two layer batt diapers, are eliminated. Moreover, the smooth contour of the batt provides better comfort and conformability for the infant than a two-piece batt diaper. The batt of the present invention provides a smooth contour at its surface rather than an abrupt change in thickness, as in a two-piece batt diaper which may produce a crease indentation in the infant's skin as his weight bears on the interface between the large and small batt. A two-piece batt diaper also tends to bend about the edges of the smaller batt as the diaper is positioned on an infant due to the uniform thickness of the larger batt and the cantilever bending effect generated therein. The contoured batt, however, bends uniformly due to its increasing thickness from the edges thus providing better conformability to the infant. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a perspective view, with certain portions broken away, of an open unfolded diaper in accordance with an embodiment of this invention; FIG. 2 is a perspective view of the double contoured cross section batt in accordance with this invention; FIG. 3 is a cross sectional view taken along plane 3--3 of FIG. 2 illustrating the longitudinal cross section contour of the batt; FIG. 4 is a cross sectional view taken along plane 4--4 of FIG. 2 illustrating the transverse contour of the absorbent batt; FIG. 5 is a schematic view of an apparatus for forming the diaper of the present invention; FIG. 6 is a plan view of two rolls of pulp board used to form the double contour cross section of the absorbent batt; FIG. 7 is a schematic cross sectional view through plane 7--7 of FIG. 5; and FIG. 7A is a schematic cross sectional view similar to FIG. 7 but illustrating another embodiment of the invention. DETAILED DESCRIPTION Referring to the drawings, and particularly to FIGS. 1 and 2, diaper assembly 10, when fully opened and laid out flat, comprises, in order, fibrous facing layer 16 adapted to be positioned adjacent the skin of an infant, absorbent fibrous panel, or batt 14, and a water-impervious sheet 12. Fibrous layer 16 is rectangular in shape, equal in dimension, and coterminous with backing sheet 12. Batt 14 comprises a panel which is double contoured, i.e. centrally contoured in the transverse and longitudinal directions to produce a smooth peak on one major surface 14a. The other major surface 14b of the batt, which may be formed by a densified layer 18, as discussed below, (FIGS. 2, 3 and 4) is planar in configuration and is in juxtaposition with the backing sheet 12. The batt 14 is rectangular in shape, but smaller than backing sheet 12 and facing layer 16, and disposed centrally thereof. The marginal portions 12a and 16a (i.e., the portions extending beyond batt 14) of sheet 12 and facing layer 16, respectively, are in face-to-face engagement with one another. Backing sheet 12 is adhered to layer 14 and 16 at the interface therebetween, as will hereinafter be described. In the preferred embodiment of the invention, moisture impervious sheet 12 is formed of polyethylene having a thickness of approximately 0.001 inch. The sheet may be smooth, or may be embossed to improve its drape and feel. Other suitable flexible moisture impervious sheets may be used in accordance with the invention, such as, for example, polyethylene terephthalate sheets having a thickness of about 0.0005 inch. Batt 14 is formed of loosely compacted short cellulose fibers, such as wood pulp fibers, or cotton linters, or mixtures thereof, which are primarily held together by interfiber bonds requiring no added adhesive, as is known in the art. Briefly, this batt is a low bulk density coherent web of loosely compacted cellulose fibers, preferably comminuted wood pulp fibers in the form of so-called "fluff". The term "short fibers," as used herein, refers to fibers less than about 1/4 inch in length, in contrast to "long fibers," or "textile length fibers," which are longer than about 1/4 inch in length, and generally are between about 1/2 and 21/2 inches in length. The former are substantially less costly than the latter. The classification of fibers by length may be carried out by the Clark Classification procedure described in the test manual of The Technical Association of Pulp and Paper Industry (TAPPI-T233 SU64). Paper-like densified layer 18 of batt 14 is formed by a slight moistening of one surface of the batt followed by the application of pressure thereto. The general nature of the batt and of its densified layer and the method of producing the same are described in U.S. Pat. No. 3,017,304, dated Jan. 16, 1962. The composite density of batt 14 including the densified layer 18 of the batt, should be above about 0.07 gm./cc., and preferably between about 0.10 and 0.15 gm./cc. The foregoing density values are applicable to the diaper as produced. In storage and handling, the loft or thickness of the batt is increased to some extent, resulting in lowered densities. Facing layer 16 is made up of a mixture of fibers consisting predominantly of short cellulosic fibers such as wood pulp fibers or cotton linters, in amounts of about 75 percent to about 98 percent, the balance being textile length fibers such as rayon. Short cellulosic fibers such as wood pulp fibers or cotton linters are substantially less expensive than textile length cellulosic fibers such as cotton and rayon, and this low cost is a factor in reducing the cost of the facing layer component of the diaper of this invention. In facing layer 16, the short fibers are in uniform admixture with 2 percent to 25 percent by weight of textile length fibers, such as 1.5 denier rayon fibers uniformly cut to 11/2 inches length. The short and long fibers are randomly and substantially uniformly dispersed and bonded with a bonding agent such as a self-cross linking acrylic emulsion. One bonding agent that has been applied with considerable success is a latex of a polyethyl-acrylate copolymer containing small amounts of acrylonitrile and a cross-linking monomer sold under the trademark HYCAR 2600 × 120. The bonding agent should be of the low viscosity type with a viscosity less than 5 centipoises. The facing layer is also treated with a wetting agent, such as, an anionic surfactant, to partially counteract the water repellency of the bonding agent and bring the facing layer to the desired degree of wettability. Typical surfactants which have been found to be suitable are the ionic sulfonated alkyl ester sold under the trademark TRITON GR-5 and the non-ionic polyoxyethylene sorbitan monolaurate sold under the trademark TWEEN 20. Facing layers of this character are described in greater detail in commonly-owned U.S. Pat. No. 3,663,348. Facing layers suitable for use in this invention have fabric weights in the range of 1 to 5 oz./yd. 2 and densities less than 0.15 gm./cc., generally in the range between 0.05 and 0.1 gm./cc. The dry strength of the facing layer, for a fabric having a weight of about 1.5 oz/yd. 2, is at least 0.15 lbs./in. of width in the machine direction and at least 0.08 lbs./in. of width in the cross direction. The fabrics have unusually good elongation, loft, softness and drape characteristics in comparison to prior products incorporating any substantial amount of short fibers. For a more detailed description of facing layers and the methods of producing them, reference may be made to the above mentioned U.S. Pat. No. 3,612,055, the disclosure of which is hereby incorporated herein in its entirety by this reference. Alternatively, the facing layer may be an apertured nonwoven fabric formed, for example, in accordance with the teachings in commonly assigned U.S. Pat. Nos. 2,862,251, 3,081,514 and 3,081,515, the disclosures of which are expressly incorporated herein by this reference. Briefly, such fabrics are foraminous structures wherein groups or groupings of fibers have been rearranged from a fibrous nonwoven starting web into positions surrounding less dense fabric portions by passage of a fluid through the starting material. The fibers within the groupings are mechanically interlocked, and may be arranged into various patterns, as is well understood by those skilled in the art. A suitable binder may be utilized to help retain the fibers in their rearranged locations, as is also well understood by those skilled in the art. The fabric can be made of naturally occurring fibers, synthetic fibers or blends thereof. Typical facing layers made of a polyester material may have a weight of 3/4 oz./yd. 2 In instances where the foramina are relatively large and particularly when the facing is formed of a polyester material, a layer of tissue or the like may be interposed between the facing layer and the batt to prevent the short paper-making fibers of the batt from sifting through the facing. It should be understood that the facing layer may also be formed of nonapertured material, such as a nonwoven isotropic web, sponge, or the like. In all of the aforementioned facings, the materials should be relatively hydrophobic so as to retard wicking within the facing layer. As is explained in U.S. Pat. No. 3,612,055, an important aspect of the improved diaper is the provision for selective wettability among the above described fibrous components, such that the moisture is selectively drawn from the facing layer into the body of the batt and then from the body of the batt into the densified layer thereof. Specifically, when liquid, such as urine, flows into a small area on the outer surface of facing layer 16, it flows preferentially into underlying batt 14 rather than to other areas of the facing layer, thus tending to restrict wetting in the facing layer to a small area and to move the liquid away from the infant's skin. When an infant's weight rests on the aforedescribed diaper construction having the double contour batt, there is a tendency for the uncompressed absorbent material of the batt 14 to be compressed by the weight. Since there is a greater thickness of material in the longitudinal and transverse central portions of the diaper than at the margins thereof, there will be greater pressure (and hence more compression) at the center. This results in a smaller effective capillary radius in the central section, and greater wickability of the more highly compressed center portion as compared to the less compressed marginal portions 14c. As a result, urine passing into the central portion of batt 14 tends to flow preferentially into the underlying portions of the batt, rather than into the marginal portions 14c of the batt. The liquid which flows through batt 14 flows preferentially into underlying densified layer 18, rather than to other areas of the loosely compacted batt, thus tending to move the liquid farther from the infant's skin. The liquid flowing into densified layer 18 tends to spread laterally because of its wicking action, and liquid which might pass through the densified layer during discharge (when flow is rapid) is held back by the impervious backing sheet for sufficient time to permit absorption to take place. Since the densified layer is confined to the central portion of the diaper, the capacity of the diaper to retain and confine liquid in this area, as compared to prior art diapers, is markedly improved. Liquid in excess of the absorptive capacity of densified layer 18 is forced back by impervious sheet 12 into the dry portion of loosely compacted batt 14, thus utilizing the additional absorptive capacity therein. It will be appreciated that liquid will initially flow into the dry portions of the relatively highly compressed central contoured portions of batt 14 before it flows into the less highly compressed marginal portions 14c thereof. The net result is that the loosely compacted marginal portions 14c act as dam-like barriers that cooperate with the densified portion 18 of batt 14 to confine liquid at the central portions of the diaper. Only after the relatively highly compressed central portions of batt 14 become saturated will liquid flow into the marginal portions 14c, and thus it will be appreciated that the diaper of the present invention effectively minimizes the likelihood that liquid will escape around the edges of the diaper. As noted above, because of the increased absorptive capacity provided by the double contoured batt construction, the aforedescribed diaper is especially adapted for use during periods of heavy discharge. In previous types of heavy duty type diapers, problems have been encountered in retaining the various batt layers in place when the diaper becomes saturated, since the increased weight attributable to the larger absorbed volume subjects the diaper to increased stresses not normally encountered in a diaper having a smaller absorptive capacity. This problem is particularly acute, since the loosely compacted fibrous layers that are conventionally used as the absorbent panel of the diaper are usually relatively flimsy and weak when compared to the facing layer and particularly to the backing layer, which ordinarily has much greater structural integrity than the other layers of the diaper. The diaper of the present invention obviates the problems noted in the preceding paragraph by having the absorbent panel and the facing layer adhered to the backing sheet substantially throughout the interface therebetween. With reference to FIG. 1, it will be noted that parallel lines of adhesive 22 are utilized to adhere the densified layer 18 or batt 14, as well as the marginal portions 16a of facing layer 16, to the backing sheet 12. Other adhesive patterns may be utilized, as will occur to those skilled in the art. In any event, since batt 14 in its entirety is secured to backing sheet 12, the batt is firmly anchored in place against movement and against disintegration. The diaper of this invention may be prepared as schematically shown in FIG. 5. Two rolls of compacted wood pulp 41a and 41b are provided to feed a source of short cellulosic fibers to grinding mill 42 from which a stream of fibers is blown downwardly through duct 42a onto belt 43 as a layer 44 weighing between about 2 and about 10 oz./yd. 2. Duct 42a is substantially rectangular in cross section, as shown in FIG. 7. To produce the contoured cross section across the web, as discussed above, two rolls of compacted wood pulp may be used. Roll 41a corresponds to the width of the batt 14 to be formed, and roll 41b is narrower than 41a to provide the transverse contour (see FIG. 4). During the grinding operation, rolls 41a and 41b are co-mingled to produce the smooth contoured cross section, as illustrated in FIG. 4. The contoured cross section across the web may also be produced by several other methods. One such method comprises feeding a source of fibers to a grinding station connected to a duct equipped with baffles at its exit to allow more fibers to be concentrated at the central portion of the web. FIG. 7a illustrates such a duct 42b and shows baffles 45 which eliminate the corners of the rectangle from the available duct area. Another method of producing a contoured cross section across the web comprises: grinding fibers at one station and depositing them to produce a continuous web at the maximum width desired and grinding fibers at another station and depositing them downstream along a band of lesser width of top of and along the median of the first-named continuous web. The longitudinal contour, as illustrated in FIG. 3, is achieved by the grinding mill by varying the speed at which the fibers from rolls 41a and 41b are deposited on belt 43. By decreasing the depository rate, the marginal areas of reduced thickness 14c are produced and, correspondingly, by increasing the depository rate the thickened central contour portion 14d is produced. Alternatively, the longitudinal contour may be achieved by (1) varying the speed of the belt segment on which the fibers are deposited, or by (2) grinding fibers at one station to produce a continuous web with a transverse contour and then sequentially grinding selected amounts of fibers at another station which are deposited on the continuous web to produce repetitive longitudinal contours. The contour thickness is preferably formed to provide a ratio of apex thickness to corner thickness in the range of 1.5 to 4. Mill 42 grinds the pulp boards into individual short fibers. However, in one preferred embodiment, some of the pulp board fibers are not completely comingled and remain joined to other fibers in small clumps, generally smaller than about 1/4 inch across. It has been found that the presence of such small clumps of fibers in the body of the batt 14 provides islands of increased tenacity for holding liquid. When an infant's weight on one portion of the batt densifies that portion, it tends to concentrate liquid in the densified portion, the presence of the clumps of fiber elsewhere in the batt tends to hold the liquid in place. Preferably from about 2 to about 10 percent of the fibers should be in the form of such clumps. The air blown contoured layer is then passed under compacting roll 46 from which it emerges with enough integrity to sustain itself as a web without the support of belt 43. The web then passes through a pair of calendar rolls 47 for further compression and then around rollers 52 and 53 which reverse the orientation of the web so that the planar surface is facing upwardly. The web then passes under nozzle 48 which deposits a fine spray of moisture on the upper surface of the web. The moistened web then passes between another set of calendar rolls 49 which exert heavy pressure on it to form a skin 18 upon its upper surface. The amount of moisture applied to the web may vary suitably from about 0.0005 to about 0.03 cc. of water per square centimeter of web surface, depending upon the thickness of the paper-like densified skin 18 desired, with lesser amounts of moisture being used for thinner webs and very thin, papery skin and greater amounts for thicker webs and skins of greater thickness. The amount of pressure applied by rolls 49 may vary from about 5 to about 100 pounds per square inch, with the commercially preferable range being from about 10 to about 50 pounds per square inch. In a typical embodiment, the web is sprayed with about 0.0015 cc. of water per square centimeter of web surface and subjected to a pressure of about 40 pounds per square inch to obtain a densified, coherent papery skin of uniform thickness on the surface of the web which has been moistened. In the absorbent web and in the batts cut therefrom, there are weak hydrogen bonds in the loosely compacted body of the batt providing sufficient strength to maintain the integrity of the batt in ordinary handling, and there are strong hydrogen bonds in the densified layer of skin to increase the cohesive strength of the composite. After the skin is formed, the absorbent web comes into contact with a web of facing material 55 and is supported thereby while being cut by cutter 56 into individual batts 14. The facing material is fed from rolls 57. Polyethylene film 12 is fed to the assembly from roll 58, lines of adhesive being applied from applicator 59. As described above, the adhesive is applied as parallel lines of beads 22 between the impervious sheet 12 and the densified layer 18 of the batt (or the facing layer in the marginal portion of the diaper). Adhesive may, if desired, by applied as a continuous layer between the polyethylene and the batt, but such application tends to provide excessive stiffness. The adhesive may also be applied in other patterns, such as spaced dots or other forms of so called "island" bonds, but fairly close overall adhesion between the sheet and the batt is required and no portion of the polyethylene should be more than about 2 inches from a point of adhesion. In the absence of such overall adhesion, polyethylene film 12 may be separated from the densified layer 18 to create substantial spaces in which uncontrollably large amounts of liquid urine can accumulate. After the facing material 16 and polyethylene 12 are brought into contact with opposite faces of the absorbent batts, the assembly is subjected to compression by rolls 60 and 61 to shape the diaper assembly, and the individual diapers are cut off by cutter 62. If desired, adhesive applicator 59 may be omitted and adhesion between the polyethylene layer and the fibrous layers may be achieved by heat sealing, employing a suitable sealing element in the production line. The diaper is normally packaged and sold in a folded condition, as is described in detail in the above mentioned U.S. Pat. No. 3,612,055. Briefly, the opposite sides of the diaper are folded inwardly toward one another, with the folded portions then being folded outwardly to provide a three-ply arrangement. The folded over portions are adhered to the main body of the diaper by centrally disposed spots of adhesive, and when it is desired to use the diaper, the folds of the diaper are opened on opposite sides of the adhesive spots, and the end portions of the diaper are placed around the waist of the infant. The overlapping corners of the end portions of the diaper are secured together by pinning, or by adhesive strips that may be attached to the backing sheet 12. It will be understood by those skilled in the art that variations and modifications of the specific embodiments described above may be employed without departing from the scope of the invention as defined in the appended claims. | Summary: A method of producing a disposable diaper and the diaper produced thereby are disclosed. The disposable multi-layer diaper includes at one side a porous fibrous facing layer to be brought into contact with an infant's skin, and includes at the other side a water impervious backing sheet, with a double contoured cross-sectional batt being interposed between the facing layer and backing sheet. The batt is smoothly contoured by increased fiber content along the transverse and longitudinal medians from the edges to the center of the batt. The batt is positioned in face-to-face engagement with the backing sheet. The batt and lateral extremities of the facing layer which extend beyond the batt are each adhered to the backing sheet. In a preferred embodiment of the invention, a paper-like, densified, highly compacted cellulosic layer is formed integrally with the batt and is positioned in face-to-face engagement with the backing sheet. The method provides a transverse peak in an air-laid web by simultaneously feeding to an individualizing station two continuous strips of compacted fibers, one strip being narrower than the other and lying along the longitudinal median of the other, the individualized fibers then being deposited on a moving foraminous belt from an air stream. Longitudinal peaks are provided by varying the rate of feed of the continuous strips to the individualizing station. | 6,994 | 308 | big_patent | en |
Summarize: The revelation that a top former commander of a Nazi SS-led military unit has lived quietly in Minneapolis for the past six decades came as a shock to those who knew 94-year-old Michael Karkoc. World War II survivors in both the U.S. and Europe harshly condemned the news and prosecutors in Poland have said they'll investigate. In this picture taken May 10, 2013, Ivan Hrushka, 69, approaches the site of a peasant house - under trees in the distance on the right - where 21 people, including nine children were burned alive on... (Associated Press) In this picture taken May 10, 2013, Heorhiy Syvyi, 78, left, and Ivan Hrushka share their war memories in their home village of Pidhaitsi close to Ukraine's western city of Lutsk. Nearly two dozen civilians,... (Associated Press) The photo taken June 3, 2013 in Chicago shows the oath of allegiance on Michael Karkoc's petition for naturalization obtained from the U.S. National Archives in Illinois. The petition was granted. Karkoc... (Associated Press) The undated reproduction shows a SS administrative file probably dated 1944 and now located in the Polish National Archive in Krakow, southern Poland. It shows a roster list for the Ukrainian Self Defense... (Associated Press) This undated reproduction shows a page of Michael Karkoc's 1949 U.S. Army intelligence file that AP had declassified by the U.S. National Archives in Maryland through a Freedom of Information Act request.... (Associated Press) The photo taken June 3, 2013 in Chicago shows the header of Michael Karkoc's petition for naturalization obtained from the U.S. National Archives in Illinois. The petition was granted. Karkoc a top... (Associated Press) The June 3, 1944 photo provided by the US Holocaust Memorial Museum shows Heinrich Himmler, centre, SS Reichsfuehrer-SS, head of the Gestapo and the Waffen-SS, and Minister of the Interior of Nazi Germany... (Associated Press) In this May 22, 1990 photo, Michael Karkoc, photographed in Lauderdale, Minn. prior to a visit to Minnesota from Soviet President Mikhail Gorbachev in early June of 1990. Karkoc a top commander whose... (Associated Press) People walk past the home in Minneapolis, Minn., where 94-year-old Michael Karkoc lives, Friday, June 14, 2013. Karkoc, a top commander of a Nazi SS-led unit accused of burning villages filled with women... (Associated Press) A man who owns the house where Michael Karkoc lived in Minneapolis said that he wasn't home, Friday, June 14 2013. Karkoc 94, a top commander of a Nazi SS-led unit accused of burning villages filled with... (Associated Press) In this picture taken May 10, 2013, a monument pays tribute to civilians who were burned alive during WWII in Pidhaitsi close to Ukraine's western city of Lutsk. The monument reads: “To our parents, wives,... (Associated Press) An Associated Press investigation found that Karkoc served as a top commander in the Ukrainian Self-Defense Legion during World War II. The unit is accused of wartime atrocities, including the burning of villages filled with women and children. "I know him personally. We talk, laugh. He takes care of his yard and walks with his wife," his next-door neighbor, Gordon Gnasdoskey, said Friday. Gnasdoskey, the grandson of a Ukrainian immigrant himself, said he was disturbed by the revelations about his longtime neighbor. "For me, this is a shock. To come to this country and take advantage of its freedoms all of these years, it blows my mind," he said. Karkoc told American authorities in 1949 that he had performed no military service during World War II, concealing his work as an officer and founding member of the legion and later as an officer in the SS Galician Division, according to records obtained by the AP through a Freedom of Information Act request. The Galician Division and a Ukrainian nationalist organization he served in were both on a secret American government blacklist of organizations whose members were forbidden from entering the United States at the time. Though records do not show that Karkoc had a direct hand in war crimes, statements from men in his unit and other documentation confirm the Ukrainian company he commanded massacred civilians, and suggest that Karkoc was at the scene of these atrocities as the company leader. Nazi SS files say he and his unit were also involved in the 1944 Warsaw Uprising, in which the Nazis brutally suppressed a Polish rebellion against German occupation. No one answered the door Friday morning at Karkoc's house on a residential street in northeast Minneapolis, where several television news trucks were parked outside. Karkoc had earlier declined to comment on his wartime service when approached by the AP, and repeated efforts to arrange an interview through his son _ including again Friday _ were unsuccessful. Late Friday, Karkoc's son, Andriy Karkos, read a statement accusing AP of defaming Karkoc, and pointed to the portion of the story about records not showing Karkoc had a direct hand in war crimes. "That's the god's honest truth," said Karkos, who uses a different spelling for his last name. "My father was never a Nazi." He said the family wouldn't comment further until it has obtained its own documents and reviewed witnesses and sources. Sam Rafowitz, an 88-year-old Jewish resident of the Minneapolis suburb of Minnetonka, grew up in Warsaw, Poland, and spent four years working in concentration camps. He took a hard line after hearing the news about Karkoc. "I think they should put him on trial," said Rafowitz, who was born near the border of Germany and Poland. He may get his wish: Polish prosecutors announced Friday they will investigate Karkoc and provide "every possible assistance" to the U.S. Department of Justice, which has used lies in immigration papers to deport dozens of suspected Nazi war criminals. Karkoc lied to American immigration officials to get into the U.S., telling authorities in 1949 that he had performed no military service during the war. He became a naturalized U.S. citizen in 1959. The AP evidence of Karkoc's wartime activities has also prompted German authorities to express interest in exploring whether there is enough to prosecute. In Germany, Nazis with "command responsibility" can be charged with war crimes even if their direct involvement in atrocities cannot be proven. Efraim Zuroff, the lead Nazi hunter at the Simon Wiesenthal Center in Jerusalem, said that based on his decades of experience pursuing Nazi war criminals, he expects that the evidence showing Karkoc lied to American officials and that his unit carried out atrocities is strong enough for deportation and war-crimes prosecution in Germany or Poland. "In America this is a relatively easy case: If he was the commander of a unit that carried out atrocities, that's a no brainer," Zuroff said. "Even in Germany... if the guy was the commander of the unit, then even if they can't show he personally pulled the trigger, he bears responsibility." Former German army officer Josef Scheungraber _ a lieutenant like Karkoc _ was convicted in Germany in 2009 on charges of murder based on circumstantial evidence that put him on the scene of a Nazi wartime massacre in Italy as the ranking officer. Prosecution in Poland may also be a possibility because most of the unit's alleged crimes were against Poles on Polish territory. But Karkoc would be unlikely to be tried in his native Ukraine, where such men are today largely seen as national heroes who fought for the country against the Soviet Union. Karkoc now lives in a modest house in an area of Minneapolis that has a significant Ukrainian population. Even at his advanced age, he came to the door without help of a cane or a walker. He would not comment on his wartime service for Nazi Germany. "I don't think I can explain," he said. Gnasdoskey said the neighborhood was once a destination for displaced persons from Slavic countries, Ukraine, Poland and other countries in the region. The area has diversified over the years, but is still occupied by the last of those residents along with some of their descendants. Karkoc and his family are longtime members of the St. Michael's and St. George's Ukrainian Orthodox Church, among several Catholic and Orthodox churches in the neighborhood. "All the time I am here, I know him as a good man, a good citizen," said the Rev. Evhen Kumka, the church's pastor. "He's well known in the congregation." Kumka moved from Ukraine to Minnesota 19 years ago to lead the congregation, and said Karkoc was already active in the church then. Kumka wouldn't say whether he'd spoken to Karkoc about his past, but said he was skeptical. "I don't think everything is correct," Kumka said. "As I know him, he is a good example for many people." Karkoc worked as a carpenter in Minneapolis, and appeared in a 1980 issue of Carpenter magazine among a group celebrating 25 years of union membership. He was a member and a secretary in the local branch of the Ukrainian National Association, a fraternal organization, and voting records obtained by the AP show he regularly voted in city, state and general elections. Members of Karkoc's unit and other witnesses have told stories of brutal attacks on civilians. One of Karkoc's men, Vasyl Malazhenski, told Soviet investigators that in 1944 the unit was directed to "liquidate all the residents" of the village of Chlaniow in a reprisal attack for the killing of a German SS officer, though he did not say who gave the order. "It was all like a trance: setting the fires, the shooting, the destroying," Malazhenski recalled, according to the 1967 statement found by the AP in the archives of Warsaw's state-run Institute of National Remembrance, which investigates and prosecutes German and Soviet crimes on Poles during and after World War II. "Later, when we were passing in file through the destroyed village," Malazhenski said, "I could see the dead bodies of the killed residents: men, women, children." Valentina Yarr of Minneapolis, a former president of the church council, said she had also known Karkoc and members of his family for many years. "I don't have anything bad to say about him, nor did I ever hear a hint of anything like this," Yarr said. "I'd rather not say anything else." In a background check by U.S. officials on April 14, 1949, Karkoc said he had never performed any military service, telling investigators that he "worked for father until 1944. Worked in labor camp from 1944 until 1945." However, in a Ukrainian-language memoir published in 1995, Karkoc states that he helped found the Ukrainian Self Defense Legion in 1943 in collaboration with the Nazis' feared SS intelligence agency, the SD, to fight on the side of Germany _ and served as a company commander in the unit, which received orders directly from the SS, through the end of the war. It was not clear why Karkoc felt safe publishing his memoir, which is available at the U.S. Library of Congress and the British Library and which the AP located online in an electronic Ukrainian library. Karkoc's name surfaced when a retired clinical pharmacologist who took up Nazi war crimes research in his free time came across it while looking into members of the SS Galician Division who emigrated to Britain. He tipped off AP when an Internet search showed an address for Karkoc in Minnesota. "Here was a chance to publicly confront a man who commanded a company alleged to be involved in the cruel murder of innocent people," said Stephen Ankier, who is based in London. The AP located Karkoc's U.S. Army intelligence file, and got it declassified by the National Archives in Maryland through a FOIA request. The Army was responsible for processing visa applications after the war under the Displaced Persons Act. The intelligence file said standard background checks with seven different agencies found no red flags that would disqualify him from entering the United States. But it also noted that it lacked key information from the Soviet side: "Verification of identity and complete establishment of applicant's reliability is not possible due to the inaccessibility of records and geographic area of applicant's former residence." Wartime documents located by the AP also confirm Karkoc's membership in the Self Defense Legion. They include a Nazi payroll sheet found in Polish archives, signed by an SS officer on Jan. 8, 1945 _ only four months before the war's end _ confirming that Karkoc was present in Krakow, Poland, to collect his salary as a member of the Self Defense Legion. Karkoc signed the document using Cyrillic letters. Karkoc, an ethnic Ukrainian, was born in the city of Lutsk in 1919, according to details he provided American officials. At the time, the area was being fought over by Ukraine, Poland and others; it ended up part of Poland until World War II. Several wartime Nazi documents note the same birth date, but say he was born in Horodok, a town in the same region. He joined the regular German army after the Nazi invasion of the Soviet Union in 1941 and fought on the Eastern Front in Ukraine and Russia, according to his memoirs, which say he was awarded an Iron Cross for bravery. He was also a member of the Ukrainian nationalist organization OUN; in 1943, he helped negotiate with the Nazis to have men drawn from its membership form the Self Defense Legion, according to his account. Initially small, it eventually numbered some 600 soldiers. The legion was dissolved and folded into the SS Galician Division in 1945; Karkoc wrote that he remained with it until the end of the war. Policy at the time of Karkoc's immigration application _ according to a declassified secret U.S. government document obtained by the AP from the National Archives _ was to deny a visa to anyone who had served in either the SS Galician Division or the OUN. The U.S. does not typically have jurisdiction to prosecute Nazi war crimes but has won more than 100 "denaturalization and removal actions" against people suspected of them. In Washington, Justice Department spokesman Michael Passman said the agency was aware of the AP story. "While we do not confirm or deny the existence of specific investigations, I can say as a general matter that the Department of Justice continues to pursue all credible allegations of participation in World War II Nazi crimes by US citizens and residents," Passman said. News of Karkoc's past also prompted anger from World War II survivors overseas, in countries where the Ukrainian Self-Defense Legion was active. In Poland, Honorata Banach told the AP she wants Karkoc to apologize. She was 20 when she fled the Polish village of Chlaniow before it was burned down by the legion. "There was so much suffering, so many orphans, so much pain," Banach said. She and her mother returned the day after the attack, she said, to see that "everything was burned down, even the fences, the trees. I could not even find my house." Survivors told her the Ukrainian legion did it, she said. Rafowitz, the survivor living near Minneapolis, said he lost his mother and other relatives at the Majadenk concentration camp in Lublin, in German-occupied Poland. He said soldiers in the camp were German but that it was run by Ukrainians. "You don't forget," Rafowitz said. "For me, it's been almost close to 70 years those things happened, but I still know about it. I still remember everything." Menachem Rosensaft, who was born in the Bergen-Belsen concentration camp, now teaches the law of genocide and war crimes at several New York universities. He said Karkoc is a reminder that the Holocaust and other genocides "cannot be viewed as abstract history." "I have every confidence that if Mr. Karkoc was not already on the Justice Department's radar screen, he now is," Rosensaft said. ___ Rising reported from Berlin, Herschaft from New York, Scislowska from Warsaw and Condon from Minneapolis. Associated Press writers Maria Danilova in Kiev, Ukraine; Efrem Lukatsky in Pidhaitsi, Ukraine; Svetlana Fedas in Lviv, Ukraine; Amy Forliti, Doug Glass and Brian Bakst in Minneapolis; and Pete Yost in Washington contributed to this report. Minnesota Nazi: US, German, and Polish authorities are now taking a look at 94-year-old Michael Karkoc’s reputed past as a Nazi commander. ‘Nazi hunters’ have had major successes and notable failures in finding and deporting Nazis. Before he successfully immigrated to the US in 1949 to settle down as a union carpenter in Minneapolis, Ukrainian-born Michael Karkoc allegedly commanded a brutal Nazi commando unit that burned Polish villages and massacred civilians at the height of Germany’s World War II offensive. If the allegations are true – and authorities in the US, Poland and Germany are now looking into the Associated Press report – Mr. Karkoc, who is in his mid-90s, could have his US citizenship revoked and be deported. If the evidence of his involvement in wartime atrocities is strong enough, he could also face war-crimes prosecution in Germany or Poland. Karkoc has not issued any public statements, and has not answered his door to reporters, according to news reports from Minneapolis. To be sure, the revelations have shocked Karkoc’s neighbors, as well as the families of World War II victims living in Minnesota and beyond. Karkoc's unit was in full operation during the 1944 Warsaw uprising, where Nazis brutally crushed Polish rebels trying to shake free from German occupation. The ability of an alleged Nazi commander to blend into US society highlights the challenges of addressing the legal and moral imperatives of the Holocaust by focusing on persecutors who tried to escape into anonymity. The question of how Karkoc was able to settle comfortably in the US – at one point appearing on the cover of a union magazine – also touches on the complex legacy of the US government “Nazi hunters” who zeroed in on hundreds of Nazi collaborators – from death camp guards turned New York housewives to the inventor of the Saturn V rocket – and whose work was hampered by political and moral questions, as well as by the difficulty of sifting through partial post-war documents, many of them hidden behind the Iron Curtain. Before being merged with another Justice Department unit in 2006, the so-called Office of Special Investigations, which opened in 1979 after a series of sensational media stories about Nazis living in the US, located 300 Nazis either in the US or trying to enter the country. | Summary: Minnesota neighbors of Michael Karkoc are stunned at the AP's finding that he led a Nazi unit said to have massacred civilians. "I know him personally. We talk, laugh. He takes care of his yard and walks with his wife," says Karkoc's next-door neighbor. Adds a pastor at a church Karkoc attends, per the AP: "All the time I am here, I know him as a good man, a good citizen. He's well known in the congregation." Karkoc's son, meanwhile, says his father "was never a Nazi": "That's the god's honest truth." US, German, and Polish prosecutors are now investigating Karkoc, the Christian Science Monitor notes. A top Nazi hunter in Jerusalem says the case should be simple for the US. "If he was the commander of a unit that carried out atrocities, that's a no brainer," he says. So why haven't US authorities already caught him, the Monitor wonders? A 2010 Justice department report says some 10,000 former Nazis may have been living in the US at one time. But "there is enormous difficulty in marshaling the evidence for these prosecutions, many subjects died before investigation was complete, the cases take years to litigate to completion, and the office (dedicated to the task) is small." | 4,395 | 305 | multi_news | en |
Summarize: Tragic: Connor Donaldson died in hospital after suffering a severe allergic reaction to a prawn balti takeaway. A 12-year-old Manchester United fan died of a severe allergic reaction to a takeaway curry despite staff assuring his family the dish contained no nuts. Moments after taking his first few bites of prawn balti, ordered from the Tyldesley Tandoori restaurant in Greater Manchester, Connor Donaldson started to gasp for breath as he suffered a severe asthma attack. His mother Sarah Donaldson, who also suffers from a nut allergy, had told staff when ordering the takeaway that her meal must not contain any trace of nuts. But investigations launched after Connor's death revealed kitchen staff had been using the same ladles and spoons to serve all food, an inquest heard. The curry was also found to contain balti paste, which contained traces of nuts. In addition the takeaway used almond powder in dishes, which contained 50 per cent peanut powder as suppliers sought to supplement the expensive ingredient with a cheaper nut. Mrs Donaldson, giving evidence at an inquest held in Bolton, said she had been diagnosed with asthma, hayfever, eczema and a nut allergy at a young age. She said while Connor had been diagnosed with asthma, hayfever and eczema, he had never formally been diagnosed with a nut allergy. 'As he had three of the four conditions I had I took it as read he would have a nut allergy and I treated him accordingly,' she said. 'I was always careful to ensure he never came into contact with any food with nuts in. He never had Chinese food and would not eat any sweets or confectionery which may contain nuts. 'From an early age he was self-disciplining and made sure he didn’t eat those products. 'Apart from a broken leg when he was nine he was happy and healthy although he would sometimes suffer from his asthma when he played football. 'They would stop the game so he could use his inhaler then start it up again.' Her son, an avid Manchester United fan, was described as 'football crazy', naming his bearded dragon Sir Alex after Sir Alex Ferguson. The inquest heard the year seven pupil was 'cheeky and funny' and 'full of life'. On October 19 last year Connor had scored the winning goal in a youth football match. When he arrived home after spending the day with friends, his mother suggested the family order an Indian takeaway. Assurances: The 12-year-old's mother Sarah Donaldson told an inquest she specifically gained assurances from staff at the Tyldesley Tandoori restaurant in Tyldesley, Greater Manchester, that the prawn balti ordered for herself and Connor would not contain any nuts, due to their allergies. Due to their nut allergies, Mrs Donaldson and her son, ordered a prawn balti after specifically gaining assurances from staff on the phone that the dish did not contain any nuts. 'The gentleman I spoke to on the phone spoke perfect English,' she told the inquest. 'We discussed that my meal couldn’t contain nuts. He assured me my meal wouldn’t contain nuts. Connor and I do not eat korma or tikka because it has nuts. 'Connor didn’t have a lot to eat - possibly only a couple of mouthfuls because it tasted funny. Within seconds I felt as though I couldn’t breathe. I took a few breaths from my inhaler and went to the front door for some fresh air. 'I came back and sat down next to Connor and he tapped me on the leg and said "I can’t breathe". 'I got his inhaler and he took ten breaths from it. The inhaler had no effect and I could tell it wasn’t going well so I straight away rang 999. 'I was comforting Connor and he slumped down and I was on the phone to the ambulance who were telling me what to do. I had already started doing CPR.' When paramedics arrived at the family's Tyldesley home, his condition was so serious there was no time to put him on a stretcher. A paramedic carried the 12-year-old to the ambulance, and continued to try and revive him for 20 minutes en route to the Royal Bolton Hospital. Shortly after arriving at the hospital doctors pronounced Connor dead. Tests confirmed he had suffered from a severe peanut allergy. He had only avoided the food on the advice of his mother, rather than seeking a professional opinion and testing. Minhaz Ahmed, the owner of the Tyldesley Takeaway, said he was satisfied serving a balti to someone with an allergy at the time - but has since realised the potentially devastating effects of cross-contamination. Football fan: The 12-year-old Manchester United fan scored the winning goal in his youth match on the day he died. Connor's family described him as 'cheeky and funny' and 'full of life' Emergency: Connor's mother Mrs Donaldson started CPR while waiting for an ambulance to arrive. Paramedics took over, attempting to revive the 12-year-old for 20 minutes en route to the Royal Bolton Hospital (pictured) where he was later pronounced dead. He maintained that woks and spoons were always clean. He said: 'I would be ok with someone having a balti with allergies. I had been aware of the risk of cross contamination. 'I know even the slightest thing can do something to another curry so we always make sure spoons and woks are clean. 'Not just for allergies but also for flavour. As far as I am concerned the same utensil should not be used in my premises if it will be used for another sauce. 'We do use Patak’s balti sauce. It says it may contain traces of nuts. On the new menu I have said that all dishes may contain nuts. I will recommend customers not to have a balti but I can’t say whether or whether it doesn’t contain nuts. Anaphylaxis is a severe, potentially life-threatening allergic reaction. Also known as anaphylactic shock, the condition can develop very rapidly. Breathing difficulties, feeling lightheaded or faint, changes to your skin including an itchy rash, and swelling body parts, particularly the face, are all signs a person is suffering a reaction. Anaphylaxis should always be treated as a medical emergency. The NHS Choices website advises that if you suspect you or somebody else is suffering an anaphylatic shock you should dial 999 immediately. Many people suffering nut allergies carry an injection of adrenaline medication with them at all times. The injections, often carried in an EpiPen, should be administered into a person's thigh muscle and held in place for 10 seconds. If the person suffering the allergic reaction falls unconscious medics advise they are put in the recovery position. If their breathing stops, CPR should be performed, the NHS Choices website states. Anaphylaxis is the result of a person's body's immune system overreacting to a harmless substance. The substances, for example nuts, are known as allergens. The condition usually develops within minutes of contact with an allergen. The most common triggers are insect stings, nuts, other types of food including milk and seafood, and certain medications. Those who suspect they maybe allergic to something should seek medical advice and tests to determine the allergen. 'There is a risk. No matter how careful you are there is a risk. There has to be. Business owners need to know because we don’t want this to happen again.' Coroner Jennifer Leeming reiterated his warning, telling the inquest that all allergy sufferers must be aware of the dangers of eating at any takeaway restaurant, especially Indians, where it is wise to assume there are traces of nuts in all dishes. Varsha Patel, an environmental health officer from Wigan Council, said an inspection of the premises found staff at the takeaway had a poor knowledge of 'cross contamination' of foods and were using the same spoons to decant different sauces. The chef was unaware Patak’s sauces had an advisory label for allergy sufferers. Miss Patel said: 'We didn’t notice any actual peanuts being used. The Patak’s paste was the only product being used which may contain traces of nuts. 'Apart from that there was almond powder and coconut flakes. The powder was being decanted into a drum which can lead to further contamination. 'Almond powder can contain peanuts because they are cheaper. 'We did some research into almond powder and there has been an adulteration with high percentages of peanut powder being used to bulk it up because it is cheaper. 'The almond powder contained at least 50 per cent peanut powder. We need to go back to the wholesaler to investigate further.' Dr Chris Moulton of Royal Bolton Hospital said: 'There are more things in Indian food than nuts capable of causing allergies. I think in this very sad incident the allergy was too overwhelming and too sudden.' The medical cause of Connor’s death was given as an acute asthma attack due to an anaphylactic reaction to food containing peanuts. Dr Vibha Sharma, an allergy expert at Royal Manchester Children’s Hospital added: 'If you are eating out in these restaurants or getting takeaways then the people providing the food need to understand you have an allergy but it is still a big risk. If due care is not taken then one would expect serious contamination.' Reaching a conclusion of accidental death, Bolton coroner Mrs Leeming said the public need to be aware of the risk that in food outlets where nuts are used there is a high likelihood of cross contamination. She added: 'Even that small amount caused by cross contamination can be dangerous to a person with a nut allergy. 'I don’t think the message is sufficiently out there that not only do you need to avoid food with nuts in them but you need to be careful about eating food from premises where nuts are used with any food stuff. Even a small amount from cross contamination can be fatal. 'What we didn’t know until the evidence of today is the risk presented by cross contamination in premises where food is sold unsealed. That would include restaurants and takeaways and cafes. 'A very small amount of allergen might be all that is required for a tragedy such as this. Depending on the individual cross contamination might be enough to result in this sort of desperate, desperate tragedy. 'I offer my sincere condolences. I am so glad he got to have that football game and scored the winning goal.' Currently, around 10 people per year die from food-related anaphylactic shock and allergic reactions in the UK | Summary: Connor Donaldson died on October 19 last year after eating a prawn balti. His mother Sarah received assurances from staff at the Tyldesley Tandoori takeaway in Greater Manchester, that the dish would not contain nuts. After eating a few mouthfuls Connor started gasping for breath. He suffered a severe asthma attack, which led to cardiac arrest. His mother told an inquest: 'He assured me my meal wouldn't contain nuts' Investigations found chefs used the same ladles and spoons for all curries. Tests also confirmed a balti paste used contained traces of nuts. Coroner Jennifer Leeming recorded a verdict of accidental death. She warned all people suffering nut allergies to avoid all takeaways. | 2,407 | 163 | cnn_dailymail | en |
Summarize: By. Steve Nolan. PUBLISHED:. 07:23 EST, 19 December 2012. |. UPDATED:. 07:59 EST, 19 December 2012. Tragic: Young mother Laura Hill, who was struggling to cope with the death of her son Jayden, pictured, was found hanged. A young mother who was struggling to come to terms with the death of her baby son has been found hanged. Friends of Laura Hill, 21, say that she was dreading Christmas without her son Jayden who died suddenly in January last year. Police confirmed that she had walked out of a hospital where she was being treated in Pembrokeshire, Wales, and was later found dead in a nearby retail park. Touching Facebook tributes were paid to the young mother today. One friend posted: 'At least you are with your baby now.' Troubled Laura had herself previously poured her heart out on her Facebook page after Jayden's death at the age of four months. She wrote: 'Miss you so much. They say times a healer but the pain is gettign worse. I would do anything to have you back. 'If I could swap places I would. Life isn't going to be the same without you. 'Can't dexcribe how much I love and miss you.' Laura, of Neyland, Pembrokeshire, gave birth to baby Jayden in 2010 and loved being a mum. She had nicknamed her baby Mooey after buying him a baby gro with a cow pattern. Laura had been admitted to a hospital in Haverfordwest for help but walked out shortly before she was found dead. A friend said: 'It is so sad - everyone knew she was struggling but we never thought it was that bad. 'A lot of people are saying she wanted. to be with her baby boy - maybe that was what was on her mind,. especially at this time of year." Loving: Proud mother Laura, left, had nicknamed baby Jayden, pictured in his cow baby gro, right, Mooey. Her friend Tara Morris said on Facebook: 'Hope you're at peace now hun, such a brave girl. At least you get to be with your lil man now.' Health chiefs confirmed today that a full investigation has been launched into Laura's death. A spokesman for the Hywel Dda Health Board spokesman said: 'A full investigation will take place. 'Due to patient confidentiality the health board cannot comment further at this time.' Investigation: Hywel Dda Health Board will examine the circumstances surrounding the death of Laura Hill, left, who friends say couldn't cope with the death of her son Jayden, pictured with Laura and right | Summary: Laura Hill, from Pembrokeshire, Wales, was found hanged at a retail park. She had walked out of a nearby hospital where she was being treated. The young mother's four month old son Jayden died suddenly last year. Friends say she was dreading the thought of Christmas without him. An investigation has been launched the Hywel Dda Health Board. For confidential support call the Samaritans in the UK on 08457 90 90 90, visit a local Samaritans branch or click here for details. | 613 | 119 | cnn_dailymail | en |
Write a title and summarize: The newly emerged mosquito-borne Zika virus poses a major public challenge due to its ability to cause significant birth defects and neurological disorders. The impact of sexual transmission is unclear but raises further concerns about virus dissemination. No specific treatment or vaccine is currently available, thus the development of a safe and effective vaccine is paramount. Here we describe a novel strategy to assemble Zika virus-like particles (VLPs) by co-expressing the structural (CprME) and non-structural (NS2B/NS3) proteins, and demonstrate their effectiveness as vaccines. VLPs are produced in a suspension culture of mammalian cells and self-assembled into particles closely resembling Zika viruses as shown by electron microscopy studies. We tested various VLP vaccines and compared them to analogous compositions of an inactivated Zika virus (In-ZIKV) used as a reference. VLP immunizations elicited high titers of antibodies, as did the In-ZIKV controls. However, in mice the VLP vaccine stimulated significantly higher virus neutralizing antibody titers than comparable formulations of the In-ZIKV vaccine. The serum neutralizing activity elicited by the VLP vaccine was enhanced using a higher VLP dose and with the addition of an adjuvant, reaching neutralizing titers greater than those detected in the serum of a patient who recovered from a Zika infection in Brazil in 2015. Discrepancies in neutralization levels between the VLP vaccine and the In-ZIKV suggest that chemical inactivation has deleterious effects on neutralizing epitopes within the E protein. This along with the inability of a VLP vaccine to cause infection makes it a preferable candidate for vaccine development. Zika fever results from an infection with the Zika virus (ZIKV), which is transmitted to humans by the bite of an infected Aedes mosquito (primarily A. aegypti). Zika virus was isolated for the first time from a Rhesus monkey in the Zika Forest in Uganda in 1947 and later from humans in Africa in 1952 [1]. The ZIKV has been transmitted in Africa for many years through a sylvatic cycle between mosquito vectors and nonhuman primates, with occasional human infections [2]. In recent years, however, epidemics of Zika have resulted from cycles of transmission between vectors and humans resulting in the spread of disease beyond the African continent into French Polynesia and other Pacific regions [3–5]. Since 2015, a dramatic spread of ZIKV that began in Brazil has taken place in South America and the Caribbean Islands with the occurrence of sporadic cases in travelers identified in the USA and Europe [6]. Currently autochthonous infections have been reported in the continental US (Florida) [7]. Infection in most cases is asymptomatic or produces a mild illness. However, contracting the virus during pregnancy is associated with birth defects, primarily microcephaly (defective brain development) as well as eye defects and hearing deficits in the infant [8]. Furthermore, an increase in cases of Guillain-Barre syndrome has been observed following ZIKV infection [9]. The seriousness of these disorders imposes a tremendous burden on public health. In addition to vector transmission, ZIKV is transmitted via sexual contact [10,11], and by body fluid [12–14]. These facts taken together with its often-asymptomatic nature makes disease control even more difficult. Zika virus is a member of the flavivirus genus within the Flaviviridae family. This family consists of a large group of enveloped viruses, which includes dengue, yellow fever, West Nile, Japanese encephalitis and others that possess a single stranded RNA genome of positive polarity, which serves as mRNA upon the infection of susceptible cells. The ZIKV RNA genome encodes one open reading frame (ORF, ~10,272 nt) and translates into a single polyprotein that similarly to other flaviviruses is co- and post-translationally cleaved by cellular and virus-encoded proteases into three structural proteins (C, prM and E) and into seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5.) that enable virus replication (Fig 1A) [15,16]. Flavivirus replication and morphogenesis occurs in close association with intracellular membranes. Nascent virions are assembled and transported through the secretory pathway and released at the cell surface. Enveloped virions are composed of a cell-derived lipid bilayer encapsulating the C-protein wrapped viral RNA genome and studded with 180 copies of the proteins E and M. During maturation within the secretory pathway the precursor prM protein is cleaved by the host cell furin protease to produce the small M protein and the fragment pr, which is released upon virus egress from the cell. The viral surface displays the E protein as the major antigenic determinant of the virus and mediates receptor binding and fusion during virus entry. Therefore, this protein is a major target for vaccine development [17]. At this time there is no approved vaccine or specific treatment available to control, combat or prevent ZIKV infection. Prophylactic vaccination represents a critical unmet need to address disease spread and its effects globally. Vaccine strategies can rely on inactivated, live attenuated, viral vectors or DNA based compositions, and some of these approaches have been recently tested for Zika vaccine development [18]. However, safety concerns may limit their potential. Here we describe a novel strategy to assemble and produce recombinant Zika virus-like particles (VLPs) and demonstrate their effectiveness when used as a vaccine in an animal model. Development of a safe and efficacious vaccine candidate will provide an effective tool to address this public health emergency. The sequences encoding the structural and non-structural genes of the Zika virus (ZIKV) were chemically synthesized by GeneArt (Life Technology) according to a specifically designed and codon-optimized GenBank sequence: KJ776791. 1, derived from Zika virus strain H/PF/2013 genome. This viral genome sequence was the most contemporary available at the time we initiated this work. The synthesized DNA fragments were subcloned into the plasmid vector pcDNA3. 4 (Life Technologies) utilizing the XbaI/EcoRV restriction enzyme sites. The non-structural genes of NS2B and viral protease NS3 were synthesized as a single codon optimized unit and subcloned into vector pcDNA3. 4 via NheI/AgeI. Plasmids were amplified in MAX Efficiency Stbl2 Competent E. Coli Cells (Life Technologies 10268–019) and purified using an EndoFree Plasmid Maxi Kit (Qiagen, MD). Cultures of Vero cells (ATCC CCL-81) were maintained in VP-SFM media (Life Technologies, CA) supplemented with 2 mM L-Glutamine, 2 mM GlutaMAX Supplement (Life Technologies, CA), 1X Non-essential amino acids solution (Life Technologies, CA), 1X ITSE (InVitria) and 500 ng/ml rhEGF (Life Technologies, CA). Expi293 cells in suspension culture were transfected with a 1: 2 ratio of pcDNA3. 4-NS2b/NS3 (ZO3) and pcDNA3. 4-CprME (ZO2) at 37°C in a 5% CO2 environment and agitated at 150 rpm. Transfected cells were harvested 72 h post-transfection and clarified via two successive centrifugations. The first clarification was performed at 400g for 10 min at 4°C followed by a second clarification at 10,000g for 10 min at 4°C. The remaining proteins in the supernatant were precipitated using 8% (w/v) of polyethylene glycol 8000 and incubated overnight at 4°C. A protein pellet was collected after centrifugation at 14,000g for 30 min at 4°C and loaded onto a 20% w/v sucrose cushion in TNE buffer (10 mM Tris-HCl, pH 8. 0,120 mM NaCl and 1 mM EDTA) and spun by ultracentrifugation at 150,000g for 2 h at 4°C. The pellet was resuspended in TNE buffer. Viruses and VLP were then purified by ultracentrifugation through a linear potassium tartrate 10–35% (w/v) / glycerol 7–30% (v/v) density gradient at 180,000g for 4 h at 4°C. Fractions were collected and analyzed by dot blot using 4G2 monoclonal mouse antibody. The 4G2 MAb reacts with dengue, Zika and other flavivirus [21] and has been shown to recognize a conformational epitope within domain II of the dengue E protein [17] and presumably recognizes a similar epitope in the Zika virus. Selected fractions were further purified and concentrated using Amicon Ultra Centrifugal Filter Unit (Millipore, MA). The cell protein content was analyzed after clarification of transfected Expi293 cells. The cell pellets were lysed with RIPA buffer (Pierce Thermo Fisher, MA) according to the vendor protocol. For Western blotting, cell lysates and concentrated culture supernatants were loaded onto a 4–12% Bis-Tris SDS-polyacrylamide gel (Life Technologies, CA). After electrophoretic separation, proteins were electro-transferred from the gel onto a 0. 45 μm nitrocellulose membrane (Life Technologies LC2001). For dot blot analysis, 3 μl of sample was applied to a 0. 45 μm nitrocellulose membrane and allowed to dry for 5 min. The nitrocellulose membranes were then blocked with 5% non-fat milk in TBST (10 mM Tris-HCl, pH 7. 4,130 mM NaCl, 2. 7 mM KCl and 0. 1% Tween-20) for 1 h at room temperature followed by overnight incubation at room temperature in primary antibody diluted with blocking buffer. Membranes were washed 3 times with 1X TBST and then incubated for 2 h with secondary antibody diluted in blocking solution. Finally, membranes were washed 3 times with 1X TBST and developed with ECL system (Life Technologies, CA). The total protein concentration was determined using the Bradford method. The E protein content in the purified VLPs and inactivated ZIKV (In-ZIKV) was determined using densitometry analysis of Coomassie blue stained SDS-PAGE gels. Different concentrations of a BSA standard were loaded onto the same gel. The stained gel image was acquired with a FluorChem M imager instrument (Protein Simple, CA). The optical density of the bands was analyzed using Alphaview software. The BSA concentrations were used to establish standard curve and parameters of the linear regression curve were used to determine E protein concentration of each VLP vaccine and In-ZIKV. Dot blot were performed with purified and quantified VLP or ZIKV and the amount of sample applied is specified in the corresponding figure legend. VLP or Zika virus samples were prepared for TEM examination as follow: Samples for immunogold labeling TEM (2. 5 μl) were loaded onto CF200-CU carbon film 200 mesh copper grid (product of Electron Microscopy Science) and incubated for 5 min at room temperature, then washed with PBS, blocked in 1% BSA in PBS for 5 min and incubated on the surface of a primary antibody drop for 30 min at room temperature. We use two antibodies, the mouse MAb 4G2 diluted 1: 500 in PBS and a polyclonal human serum from a Zika virus infected patient diluted 1: 20 dilution in PBS. The grids were then washed 6 times with PBS and incubated on the surface of a drop of secondary antibody conjugated with gold beads for 30 min. A goat anti-mouse antibody conjugated with 10 nm gold beads was used with the mouse MAb 4G2 and a goat anti-human antibody conjugated with 6 nm gold beads was used with the human serum. Grids were finally washed 6 times with PBS, and then fixed for 15 min with 4% paraformaldehyde in PBS, washed with PBS and subsequently washed twice with 0. 2M Sodium cacodylate buffer and finally stained with 1% uranyl acetate solution. The aim of the study was to determine the immunogenicity and efficacy of a VLP-based Zika vaccine and its capacity to elicit a strong neutralizing serum protective immune response. Experimental groups comprised eight mice (n = 8) in order to study pairs of subjects while being able to reject a null hypothesis with statistical power of >99% and a standard deviation of 19. The alpha (α) error probability associated with this test is 0. 01. Nine groups of 6 to 8-week old female BALB/c mice (n = 8) were inoculated twice (day 0 and day 24) via the intramuscular (IM) route with either VLP vaccine, formalin inactivated ZIKV MR-766 (In-ZIKV) control or PBS plus adjuvant negative control (Neg. Ctr.). Mice received doses of either 1 μg or 4 μg of total E protein content formulated alone or admixed in a 1: 1 volume ratio with a squalene-based oil-in-water nano-emulsion AddaVax (InvivoGen, CA). Serum samples were collected from all animals at day 42. Serum IgG titers against Zika viruses were determined by ELISA. Briefly, assays were performed in 96-well plates coated with 50 μl/well of purified and inactivated Zika virus MR-766 or FSS-13025 (2 μg/ml total protein concentration) at 4°C overnight. Subsequently, plates were washed 3 times with PBS-T (0. 05% Tween-20 in phosphate buffered saline) and then blocked with 100 μl of blocking solution (5% non-fat milk in PBS-T) for 1 h at room temperature (RT). Vaccine and control sera were diluted in fourfold series in blocking buffer and applied to each well in triplicate and incubated for 2 h at RT. After 6 washes with PBS-T, plates were incubated for 2 h at RT with 50 μl of HRP-conjugated goat anti-mouse IgG (H+L) secondary antibody diluted (1: 5000) in blocking buffer). Subsequently, plates were washed 6 times with PBS-T and developed by adding 50 μl per well of Ultra TMB solution (Thermo Scientific, MA) for 20 min of incubation and stopped with 100 μl of stop solution (2 M HCL solution). ELISA end-point titer for each group was calculated as the reciprocal of the highest dilution with OD value 2σ above the mean of the negative control wells. The plaque reduction neutralization test was carried out in Vero cells using sera from immunized mice and either MR-766, or FSS-13025 Zika viruses following the method described for the evaluation of flavivirus vaccine efficacy [22,23]. Briefly, ZIKV was amplified and titrated in Vero cells using the same plaque visualization procedure described below. Vero cells were seeded in 24-well plates at a density of 6x104 cells per well 24 h prior to the initiation of the test. Control and test sera were heat inactivated at 56°C for 30 min. Starting at a 1: 20 dilution, the sera were two-fold serially diluted with cell culture media supplemented with penicillin and streptomycin. Equal volumes of diluted virus to form ~60 plaques per well were added to each serum dilution. The sera-virus mixtures were incubated for 1 h at 37°C in a 5% CO2 environment. Afterwards, each dilution was applied in duplicated to wells of 85% confluent monolayer of Vero cells and incubated for 90 min at 37°C in a 5% CO2 incubator. Thereafter, the inocula were removed and a 1 ml overlay of 1. 8% carboxymethyl cellulose (CMC) in culture medium was added to each well. Plates were then incubated for 3 days at which time the CMC overlay was removed, cells washed with PBST (phosphate buffer saline plus 0. 05% Tween 20) and fixed with cold 80% acetone for 10 min at -20°C. Subsequently, plates were washed with PBST and then incubated with blocking buffer (2. 5% non-fat milk in 0. 5% of Triton X-100 PBS solution) for 1 h in a 37°C incubator. After one wash, the primary antibody (MAb 4G2) diluted 1: 500 in blocking buffer was applied for 2 h at RT, followed by two PBST washes and incubation for 1 h at RT with a goat anti-mouse AP conjugated secondary antibody diluted (1: 2000) in blocking buffer. Finally, plates were washed twice with PBST and once with alkaline phosphate buffer (APB: 100 mM Tris-HCl pH9. 0,150 mM NaCl, 1 mM MgCl2). Viral plaques were detected by adding the alkaline phosphate (AP) substrate nitro blue tetrazolium chloride (NTB) and 5-brome-4-chlore-3-indolyl phosphate (BCIP) prepared and used as follows: Combine 33 μl of NTB (50 mg/ml in 70% dimethylformamide) with 5 ml of APB mix well and then add 16. 5 μl of BCIP (50 mg/ml in 100% dimethylformamide) and the mixture should be used within 1 h of preparation. Plaques were counted and PRNT50s were determined using the PROBIT method [24]. The neutralization power calculated is expressed as the reciprocal of the highest serum dilution that neutralizes 50% of the virus and the histograms represent the average neutralization 50% per group and their standard deviations. Suspension cultures of U937 cells grown in RPMI supplemented with 10% of FBS were used in the ADE assay, which was performed according to Diamond et. at. [25]. Prior to the initiation of the experiment (72h), the U397 cells were differentiated toward the granulocyte or macrophage lineage by the addition of dimethyl sulfoxide (DMSO; 1. 25%) [25]. DENV-2 virus (50μl) was mixed with diluted serum samples (four for each vaccine group) and controls (virus alone or 4G2 MAb-200ng) and then added to 2. 5x105 cells resulting in a multiplicity of infection of 3 (MOI: 3). The resulting mixture of cells, virus and serum was incubated for 2 h at 37°C. Subsequently, cells were washed four times to remove free virus, suspended in culture medium and then incubated for 96 h at 37°C. Thereafter, virus titers in the culture supernatants were determined by plaque assay as described above in the PRNT assay. All statistical calculations were carried out using GraphPad Prism 4 software. Tests between two groups used two-tailed Student’s t-test. The P-value was calculated for <0. 05 level of significance. Mouse studies were carried out in the Department of Comparative Medicine, Animal Facility of the New York Medical College, Valhalla, NY. All animals were cared for in compliance with the Guide for the Care and Use of Laboratory Animals and all experiments were approved by the New York Medical College’s IACUC. Mice were housed in an AAALAC-accredited facility. Early studies on flavivirus replication have demonstrated that together with complete infectious virions, particles are also produced that lack the viral RNA genome, which have been termed small, non infectious subviral particles (SVPs) or virus-like particles (VLPs) [26]. Assembly of these non-infectious particles using recombinant methods has been utilized to study protein function, morphogenesis and structure [27–29] and to generate flaviviruses vaccines, which have proven to be immunogenic [30]. We have found a new and effective strategy to assemble ZIKA VLPs and utilize them for vaccine development. Formation of VLP is accomplished by the co-expression of the Zika virus structural proteins CprME together with a truncated form of the protease NS3Pro linked to its cofactor NS2B constituting the viral NS2B/NS3Pro protease complex (Fig 1B). Transfection of suspension cultures of Expi-293 cells with plasmids expressing CprME and NS2B/NS3Pro produces the structural polypeptide CprME, which undergoes proteolytic cleavages mediated by cellular and the co-expressed NS2B/NS3Pro proteases (Fig 1B). Expression of NS2B/NS3Pro and processing of the polyprotein CprME was evaluated by Western blot analysis using lysates of cells transfected with a single plasmid expressing CprME (ZO2) or a single plasmid expressing NS2B/NS3Pro (ZO3) or a combination of both plasmids. This analysis showed that the protease NS2B/NS3Pro was expressed and self-cleaved rendering NS2B (~14KDa) (Fig 2D) and NS3Pro (~19KDa) (Fig 2C) when cells were transfected with the plasmid ZO3 alone (expressing NS2B/NS3Pro) or together with ZO2 (expressing CprME) but not in cells only transfected with the plasmid ZO2 (CprME) (Fig 2C and 2D). Analysis of a ZIKV infected Vero cell lysate revealed the expression of the full-length NS3 protein (containing both the protease and the helicase domains), which was absent from the lysate of uninfected (mock) Vero cells (Fig 2C). Similarly, Western blot studies demonstrated that the polyprotein CprME was expressed and processed yielding the expected proteins E and prM, as well as intermediate cleavage products including prM and CprM (Fig 2A and 2B). Release of prM and C from ZO2 alone seems to occur at a lower frequency in the absence of the virus protease complex NS2B/NS3Pro. Therefore to detect M and pr in ZO2 alone requires higher amount of loaded material than the one used (Fig 2B). We were unable to detect the C protein in neither the ZIKV infected cell lysate nor the VLP transfected lysates using the currently available anti-C antibody. However, the detection of E and prM suggests that C is cleaved as well. This analysis demonstrates protein expression as well as the activity of NS2B/NS3Pro, which is able to self-cleave uncoupling NS2B and NS3Pro before subsequently cutting of C from the polyprotein (Figs 1 and 2A–2D). Further cleavage processing steps mediated by cellular signalase proteases separate E from M and C from pr. The released E protein shows slightly greater size than the E of Zika MR-766 virus infected Vero cells (Fig 2A), due to the lack in this ZIKV strain of four amino acids in the E protein (153–156) including the glycosylation site asparagine (N-154), which are present in the E protein of ZIKV H/PF/2013 used for VLP production as well as other ZIKV strains such as the more contemporary strain FSS-13025 used as control [31]. At the time of immunization, only the viral strain MR-766 was available. The recombinant production of Zika VLPs was carried out in suspension cultures of Expi-HEK293 cells following co-transfection of the plasmids ZO2 (CprME) and ZO3 (NS2B/NS3 Pro). The VLPs were harvested from the culture supernatant and purified as described in Material and Methods. The Zika virus grown in Vero cells was subjected to an analogous purification scheme as the one described for the VLPs. The purity and identity of the protein component of the purified VLP and Zika virus were assessed by Coomassie blue staining and Western blot analysis. As shown in Fig 3A, the E protein was readily detected as the predominant component of both the ZIKV and the VLP preparations. The identity of the E protein was verified by Western blot analysis and exactly correlated with the E protein visualized in the stained gel (Fig 3B). The migration difference of the E proteins detected in the cell lysates was also seen between the E protein of the purified ZIKV-MR-766 and VLPs (Fig 3A and 3B). This disparity, as noted above, is due to the lack of four amino acids including the glycosylation site N-154 in ZIKV-MR-766. These residues are present in ZIKV-FSS-13025 and H/PF/2013 and sequences of this strain were used to produce the VLPs. Examination by Western blot of the migration patterns of the E protein from ZIKV-MR766, FSS-13025 and VLPs (genes derived from H/PF/2013) showed that the MR-766 E protein migrated slightly faster than the E protein from ZIKV FSS-13025 and VLPs, which exhibit similar size (Fig 3C). Coomassie staining also showed minor unidentified proteins in the VLP preparation that were also present in the ZIKV albeit in lesser amounts (Fig 3A). The M protein, a major structural component of the Zika virus, was clearly identified by Western blot analysis in both the ZIKV and VLP preparations but it was not clearly seen in the stained gel, possibly due to its small size and low concentration, which precluded retention of sufficient amounts of bound dye for clear detection (Fig 3A). Both, E and M the major structural proteins were clearly identified by Western blot (Fig 3B). Furthermore, to corroborate whether conformational neutralizing epitopes of the E protein were exhibited on the VLPs, we probed by dot blot purified VLPs and ZIKVs with the domain specific neutralizing MAbs, ZV-48, ZV-64 and ZV-67 [20] in addition to the mouse MAb 4G2. This examination showed that the VLPs were indeed recognized by these MAbs indicating that these neutralizing epitopes were properly displayed in the VLPs as they also were in the ZIKV MR-766 and FSS-13025 (Fig 4). We have also observed that the reactivity of Zika viruses with these MAbs is mostly abrogated after the viruses are inactivated with formaldehyde (Fig 4). The VLP sample remained untreated. To further characterize the structure and surface composition of the VLPs, we examined purified material by transmission electron microscopy (TEM). Negative staining studies revealed that the VLPs are fairly homogeneous spherical structures with an average diameter of 60 nm. The VLP surfaces appeared smooth without noticeable projection or rough features. Comparative analysis with purified ZIKV showed that both VLP and virus particles are similar in size, morphology and surface appearance (Fig 5A, 5B and 5E). This is in agreement with recent reports of the Zika virus structure [31,32]. To better define the surface composition of the VLPs, we probed the recombinant particles by immunogold labeling with two distinct antibodies, 4G2, a MAb that recognizes a conformational loop in domain II of the E protein shared by some members of the flavivirus family including Zika [21], and a polyclonal human serum from a Zika patient. Subsequently these preparations were examined by negative staining TEM. These studies demonstrated the reactivity of the VLPs with both antibodies (Fig 5C and 5D) indicating not only that the E protein is displayed on the surface of the particles but that it also maintains its native conformation based on reactivity with the MAb 4G2 (Fig 5C). Examination of purified ZIKV also showed equivalent reactivity with the 4G2 antibody (Fig 5F) confirming that both the VLP and live ZIKV exhibit on their surfaces the conformational site recognized by 4G2 (Fig 5C and 5F). We further probed VLPs with the serum from a Zika patient and found that it also binds to the particles’ surface providing addition evidence that the ZIKV major surface glycoprotein E is indeed present on the VLP surface (Fig 5D). We sought to assemble Zika VLPs with the main objective of developing a safe and effective vaccine to stem the rapidly spreading Zika epidemic. To establish immunogenicity and in-vitro efficacy of a VLP-based Zika vaccine, we designed a mouse study to assess the performance of a VLP vaccine and to compare the outcome to an equivalent formulation and dose of an inactivated Zika virus (In-ZIKV) control. Given the urgency of advancing a Zika vaccine and the lack, at the onset of the study, of well-established Zika animal models, we selected a mouse model to carry out this vaccine evaluation. Two groups of 6 to 8- week old female Balb/c mice (n = 8 each) were immunized via the intramuscular route (IM) twice, three weeks apart, with two difference doses, 1 μg and 4 μg of total E protein of VLP vaccine formulated with or without adjuvant (AddaVax, InVivoGen) a squalene-oil-in water nano-emulsion that stimulates a balanced immune response and has a formulation similar to MF59, a licensed adjuvant in flu vaccines in Europe [33,34]. Similar groups of mice (n = 8 each) were immunized via the same route and schedule with a formalin inactivated Zika virus (In-ZIKV) control at the doses of either 1 μg or 4 μg of total E protein content also formulated with or without adjuvant. A negative control (Neg. Ctr.) group (n = 8) received phosphate buffered saline (PBS) plus adjuvant following the same immunization regimen as used for the vaccine groups. Three weeks after the booster immunization, animals in all groups were terminally bled and sera samples prepared for immunogenicity and efficacy evaluation. We assessed the level of the total serum IgG response in all vaccinated animals by ELISA using as antigens two Zika virus strains, one isolated in 1947 (Zika virus MR-766) and a second more current strain isolated in Cambodia in 2013 (Zika virus, FSS 13025). The ELISA results demonstrated that mice vaccinated with the low dose (1 μg) or high dose (4 μg) of VLP vaccine stimulated the production of high levels of serum antibodies against both Zika virus strains (Fig 6 Upper and Lower panels). This response was enhanced when the VLP vaccines were formulated with adjuvant. The 1 μg VLP vaccine plus adjuvant elicited a markedly increased level of serum IgG as compared to VLP alone. This enhancement was even more significant using the 4 μg VLP vaccine dose formulated with adjuvant. Similar results were obtained with the two Zika virus antigens used in the ELISA studies (Fig 6 Upper and Lower panels). Mice vaccinated with the inactivated Zika virus (In-ZIKV) produced a serum IgG response that was quite comparable to the one triggered by the VLP vaccines. Clearly, both the VLP and the inactivated Zika virus control compositions were capable of eliciting a high antibody response against two Zika viruses. In contrast, the negative control group did not demonstrate a specific IgG response against Zika. Elicitation of high titers of specific neutralizing antibodies has been found to correlate with protection against several flaviviruses including yellow fever (YF) tick-borne encephalitis, dengue and Japanese encephalitis [35–37]. Although this has not yet been established for Zika it is very likely that the presence of high titers of neutralizing antibodies confer protection to infection. Therefore, we assessed the level of neutralizing antibodies elicited by the VLP vaccine and controls using a plaque reduction neutralization test (PRNT) with the two Zika viruses previously described. This evaluation with the ZIKV MR-766 showed that the VLP vaccine at the lower dose (1 μg) elicited neutralizing antibody titers significantly higher than the negative control group, which corresponds to a PRNT50: 1085 for the vaccine versus PRNT50: <25 for the negative control (Fig 7 Left panel and Table 1). This response was enhanced when adjuvant was part of the formulation raising the PRNT50: 1301. In contrast, the inactivated Zika virus (In-ZIKV) control at equivalent dose and formulation stimulated much lower neutralizing titers than the VLP vaccine reaching PRNT50: 79 for the 1 μg dose and PRNT50: 794 for the 1 μg plus adjuvant formulation. Increasing the VLP vaccine dose to 4 μg with or without adjuvant significantly raised the neutralizing antibody titers to PRNT50: 2978 and PRNT50: 20854, respectively. Including the adjuvant in the 4 μg VLP vaccine formulation heightened neutralization titers seven fold. The In-ZIKV high dose (4 μg) control showed improved titers when adjuvant was added PRNT50: 1408 versus PRNT50: 430 for In-ZIKV alone, but neither formulation gives rise to the neutralization activity attained with the VLP vaccine. All VLP vaccine formulations elicited statistically significant higher neutralizing antibodies titers than the equivalent compositions of the inactivated Zika virus vaccine with the exception of the 1 μg plus adjuvant dose, where the VLP induced higher titers but the difference was not significant. Neutralization analysis with the more contemporaneous ZIKV FSS-13025 resulted in a neutralization pattern that resembled the one seen with ZIKV MR-766. The VLP vaccine showed significantly higher neutralizing titers against ZIKV FSS-13025 than the In-ZIKV vaccine and this increased with the rise in dose and the incorporation of adjuvant (Fig 7 Right panel and Table 1). Furthermore, we used as a reference in the PRNT50 a human serum from patient who had recovered from a Zika infection. This revealed high titers of neutralizing antibody against the two Zika viruses tested; although the neutralizing activity was 4-fold higher for ZIKV-FSS virus (PRNT50: 17,067 for FSS versus and PRNT50: 4267 for MR-766) (Fig 7 Left and Right panels and Table 1). Since the human serum was obtained from a recently infected patient, it is likely that it better neutralized the more contemporaneous ZIKV–FSS suggesting that differences in neutralizing epitopes may exist between these two viruses. Given the fact that until now we have had access to only one Zika infected human serum sample, we could not perform a more comprehensive comparison with the VLP vaccine. Nonetheless, these results showed that the VLP vaccine was superior in eliciting neutralizing antibody responses than equivalent compositions of an In-ZIKV control (Fig 7 Left and Right panels and Table 1). Considering that flavivirus infections may induce antibody-dependent enhancement of infection (ADE) with heterologous/co-circulating viruses, we tested whether the antibody response elicited by the VLP or controls vaccination against ZIKV may play a role in augmenting dengue virus infection. Here, we utilized an in vitro ADE assay [25] and measured whether DENV-2 infection was enhanced in the presence of anti-ZIKV antibodies. We used the MAb 4G2, which enhances dengue virus infections as positive control and DENV-2 alone as reference. This study showed that the antibody response to ZIKV elicited by VLP vaccination did not enhance DENV-2 infection (Fig 8). Nor did the sera from the In-ZIKV and the negative control vaccination when compared to DENV-2 with 4G2 MAb, which serves as the positive control and significantly enhanced DENV-2 infection (Fig 8). These data suggest that the antibody response elicited by these ZIKV vaccines at the dilution tested in the assay did not induce ADE of dengue 2-virus infection. The ongoing Zika epidemic has already resulted in over 1500 cases of microcephaly in Brazil and 15 cases in the continental United States born to mothers who had traveled to affected areas [38,39]. Many other countries in South America and the Caribbean are also experiencing the effects of the Zika epidemics and some autochthonous infections have been reported in the continental US in Florida. In addition to the mosquito vector direct infection there appear to be other forms of transmission including via sexual contact [11,13] potentially increasing the risk of viral dissemination. Developing a safe and effective vaccine to control and combat the spread of the Zika virus is a high priority. Our work describes a new strategy to generate Zika virus-like particles (VLPs), providing a potentially safe and effective platform for the rapid production of a candidate for clinical development of a prophylactic Zika vaccine. The single co-expression of the structural polyprotein CprME together with the non-structural NS2B/NS3Pro suffices for the processing of CprME leading to the self-assembly and release of VLPs devoid of viral RNA. Purified VLPs resemble the wild type Zika virus in size, morphology and antigenic composition offering a suitable strategy for vaccine development. Several highly effective viral vaccines are based on the VLP strategy [40,41] providing rationale for the use of Zika VLP for vaccine development. VLP vaccine production can be attained in suspension cultures of mammalian cells using standard fermentation technology. Considering that VLPs are not infectious or able to replicate, inactivation is not required better preserving protein structure and conformational epitopes. Our results show that all VLP vaccine and In-ZIKV control formulations elicited high titers of serum IgG antibodies. However these high levels of Zika specific antibody did not correlate, in the In-ZIKV vaccine control, with induction of high titers of neutralizing antibodies, which are the benchmark of protection. Most of our VLP vaccine formulations stimulated neutralizing antibody titers that were significantly higher than those induced by the In-ZIKV control. The discrepancy between high ELISA titers and low neutralization response in the In-ZIKV control, as compared to the VLP vaccine, may be attributed to the distortion of critical neutralizing epitopes as a result of ZIKV inactivation, which may reduce the repertoire of neutralizing sites. We have seen some of these effects where formalin inactivation of the Zika viruses abrogated most of the reactivity with the MAbs 4G2, ZV-48, ZV-64 and ZV-67, which recognize conformational epitopes in the fusion loop of domain II and domain III of the E protein (Fig 4). Therefore, the display of native and properly configured proteins on the surfaces of the VLPs appears to exhibit to the immune system a better antigenic array, which promotes the stimulation of a more diverse neutralizing response as we have seen with these VLP vaccine formulations. Suboptimal neutralizing antibody levels may contribute to antibody-enhancement of disease in dengue, which by phylogenetic analysis is closer to Zika than other flaviviruses. In fact, one study has shown that combining dengue cross-reacting antibodies with Zika virus enhanced Zika infection in a cell culture assay [42]. In recent years, quaternary epitopes identified on the surface of dengue have been recognized as strong neutralizing sites [43–46]. Furthermore, a recent study has shown that monoclonal antibodies recognizing these highly neutralizing epitopes of dengue have also shown protection against Zika highlighting the significance of these types of epitopes in providing protection against Zika virus infection [47]. Although the correlates of protection for a Zika vaccine remain to be established, the elicitation of a strong and diverse neutralizing response may greatly contribute to protection against Zika infection. As shown in this study, the VLP based Zika vaccine elicited a robust neutralizing antibody response suggesting that critical conformational epitopes were indeed displayed for effective immune stimulation. In contrast to the response elicited by our VLPs, the In-ZIKV produced a much-reduced neutralizing response, even though the ELISA titers were as high as those induced by the VLP vaccine, suggesting that important neutralizing epitopes were not properly displayed in the In-ZIKV composition. To investigate the possibility that the anti-ZIKV antibodies elicited by VLP vaccination may partially bind to dengue virus and contribute to antibody-dependent enhancement (ADE) of dengue infection, we tested diluted serum samples of VLP and control immunized animals in an ADE in vitro assay. This evaluation demonstrated lack of enhancement of DENV-2 infection when either the VLP vaccine or control immunizations stimulated antibodies were tested. In contrast, the 4G2 MAb significantly enhanced dengue infection, a property well established for this antibody [42]. This outcome provides some indication that ZIKV VLP vaccination does not appear to mediate ADE at least with DENV-2. This study describes a safe, effective and straightforward strategy to rapidly produce a Zika vaccine. VLPs are produced in mammalian suspension cell cultures offering a suitable system for rapid scale up of manufacturing, without the risk of working with an infectious agent. The lack of infectivity of the product eliminates the need for chemical inactivation, which may compromise vaccine efficacy and safety. The particulate nature of the vaccine and the preservation of a variety of conformational antigenic sites may even render this vaccine efficacious in humans without using adjuvant if its incorporation into a vaccine raises safety concerns. In summary, the Zika VLP platform puts forward a vaccine composition and production system ready for clinical development of a safe and effective prophylactic Zika vaccine, which is greatly needed to meet the challenges imposed by the spread of the Zika epidemic. | Title: Zika virus-like particle (VLP) based vaccine Summary: Zika is an emerging mosquito-borne infection for which vaccines or specific treatments are not available to combat and control its rapid dissemination and deleterious effects. This work describes a novel strategy for the development of a virus-like particle (VLP) based Zika vaccine and shows its effectiveness when tested in a murine model. VLPs are non-infections mimics of the Zika virus, thus chemical inactivation is not required. A VLP vaccine is able to maintain native epitopes structures and to perform better than an inactivated Zika virus control. Our results show feasibility for the rapid development of a safe and potentially highly effective VLP based Zika vaccine for humans. | 10,001 | 160 | lay_plos | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Benefit Authors without Limiting Advancement or Net Consumer Expectations (BALANCE) Act of 2003''. SEC. 2. FINDINGS. The Congress makes the following findings: (1) The law of copyright is often described as a ``difficult balance between the interests of authors... in the control and exploitation of their writings... on the one hand, and society's competing interest in the free flow of ideas, information, and commerce on the other hand.'' Sony Corp. v. Universal City Studios, Inc., 464 U.S. 417, 429 (1984). (2) Copyright seeks to encourage and reward creative efforts by securing a fair return for an author's labor. Twentieth Century Music Corp. v. Aiken, 422 U.S. 151, 156 (1975). At the same time, ``[f]rom the infancy of copyright protection, some opportunity for fair use of copyrighted materials has been thought necessary to fulfill copyright's very purpose, `[t]o promote the Progress of Science and useful Arts...''' Campbell v. Acuff-Rose Music, Inc., 510 U.S. 569, 575 (1994). (3) ``[P]rivate motivation must ultimately serve the cause of promoting broad public availability of literature, music, and the other arts... When technological change has rendered its literal terms ambiguous, the Copyright Act must be construed in light of this basic purpose.'' Twentieth Century Music Corp., 422 U.S. at 156. (4) Advances in technology have often prompted changes to the copyright laws to maintain the balance. For example, the development of player pianos preceded the enactment of the Copyright Act of 1909. The development of cable television prompted complex reforms to section 111 of title 17, United States Code. Sony, 464 U.S. at 430-31. (5) The development of digital technology and the rise of the Internet have once again altered the balance. On the one hand, digital technology threatens the rights of copyright holders. Perfect digital copies of songs and movies can be publicly transmitted, without authorization, to thousands of people at little or no cost. On the other hand, technological control measures give copyright holders the capacity to limit nonpublic performances and threaten society's interests in the free flow of ideas, information, and commerce. (6) The Digital Millennium Copyright Act (``DMCA'') was enacted as an attempt to safeguard the traditional balance in the face of these new challenges. It gave copyright holders the ability to fight digital piracy by employing technical restrictions that prevent unlawful access and copying. In practice, however, the DMCA also endangered the rights and expectations of legitimate consumers. (7) Contrary to the intent of Congress, section 1201 of title 17, United States Code, has been interpreted to prohibit all users--even lawful ones--from circumventing technical restrictions for any reason. As a result, the lawful consumer cannot legally circumvent technological restrictions, even if he or she is simply trying to exercise a fair use or to utilize the work on a different digital media device. See, e.g., Universal City Studios, Inc. v. Reimerdes, 111 F. Supp. 2d 294, 321-24 (S.D.N.Y. 2000) (DMCA failed to give consumers the technical means to make fair uses of encrypted copyrighted works). (8) The authors of the DMCA never intended to create such a dramatic shift in the balance. As the report of the Committee of the Judiciary of the House of Representatives accompanying the DMCA stated: ``[A]n individual [should] not be able to circumvent in order to gain unauthorized access to a work, but [should] be able to do so in order to make fair use of a work which he or she has acquired lawfully.'' House Report 105-551, Part I, Section-by-Section Analysis of section 1201(a)(1). (9) It is now necessary to restore the traditional balance between copyright holders and society, as intended by the 105th Congress. Copyright laws in the digital age must prevent and punish digital pirates without treating every consumer as one. SEC. 3. PROTECTING FAIR USE AND CONSUMER EXPECTATIONS IN THE DIGITAL WORLD. (a) Fair Use.--The first sentence of section 107 of title 17, United States Code, is amended by inserting after ``or by any other means specified in that section,'' the following: ``including by analog or digital transmissions,''. (b) Permissible Uses of Digital Works.-- (1) In general.--Chapter 1 of title 17, United States Code, is amended by adding after section 122 the following: ``Sec. 123. Limitations on exclusive rights; Permissible uses of digital works ``(a) Use of Lawfully Obtained Digital Works.--Notwithstanding the provisions of section 106, it is not an infringement of copyright for a person who lawfully obtains a copy or phonorecord of a digital work, or who lawfully receives a transmission of a digital work, to reproduce, store, adapt, or access the digital work-- ``(1) for archival purposes, if all such archival copies are destroyed or rendered permanently inaccessible in the event that continued possession of the work should cease to be rightful; and ``(2) in order to perform or display the work, or an adaptation of the work, on a digital media device, if the work is not so performed or displayed publicly. ``(b) Effect of Licenses.--When a digital work is distributed to the public subject to nonnegotiable license terms, such terms shall not be enforceable under the common laws or statutes of any State to the extent that they restrict or limit any of the limitations on exclusive rights under this title. ``(c) Definitions.--As used in this section, the following terms have the following meanings: ``(1) A `digital work' is any literary work (except a computer program), sound recording, musical work, dramatic work, or motion picture or other audiovisual work, in whole or in part in a digital or other nonanalog format. ``(2) A `digital media device' is any hardware or software that converts copyrighted works in digital form into a format whereby the images and sounds are visible or audible, or retrieves or accesses copyrighted works in digital format and transfers or makes available for transfer such works to such hardware or software. ``(d) Construction.--Nothing in this section shall enlarge or diminish any of the other limitations on exclusive rights contained in this title, including any limitations that relate to archival activities by a library or an archives under sections 107 and 108.''. (2) Conforming amendment.--The table of sections for chapter 1 of title 17, United States Code, is amended by adding at the end the following new item: ``123. Limitations on exclusive rights; Permissible uses of digital works.''. SEC. 4. DIGITAL FIRST SALE. Section 109 of title 17, United States Code, is amended by adding at the end the following: ``(f) The privileges prescribed by subsections (a) and (c) apply in a case in which the owner of a particular copy or phonorecord of a work in a digital or other nonanalog format, or any person authorized by such owner, sells or otherwise disposes of the work by means of a transmission to a single recipient, if the owner does not retain the copy or phonorecord in a retrievable form and the work is so sold or otherwise disposed of in its original format.''. SEC. 5. PERMISSIBLE CIRCUMVENTION TO ENABLE FAIR USE AND CONSUMER EXPECTATIONS. Section 1201 of title 17, United States Code, is amended-- (1) by redesignating subsections (c) through (k) as subsections (d) through (l), respectively; and (2) by inserting after subsection (b) the following: ``(c) Circumvention for Noninfringing Uses.--(1) Notwithstanding any other provision in this title, a person who lawfully obtains a copy or phonorecord of a work, or who lawfully receives a transmission of a work, may circumvent a technological measure that effectively controls access to the work or protects a right of the copyright holder under this title if-- ``(A) such act is necessary to make a noninfringing use of the work under this title; and ``(B) the copyright owner fails to make publicly available the necessary means to make such noninfringing use without additional cost or burden to such person. ``(2) Notwithstanding the provisions of subsections (a)(2) and (b), any person may manufacture, import, offer to the public, provide, or otherwise make available technological means to circumvent a technological measure that effectively controls access to a work protected under this title or protects a right of a copyright holder under this title, if-- ``(A) such means are necessary to make a noninfringing use under paragraph (1)(A); ``(B) such means are designed, produced, and marketed to make a noninfringing use under paragraph (1)(A); and ``(C) the copyright owner fails to make available the necessary means referred to in paragraph (1)(B).''. | Title: To amend title 17, United States Code, to safeguard the rights and expectations of consumers who lawfully obtain digital entertainment Summary: Benefit Authors without Limiting Advancement or Net Consumer Expectations (BALANCE) Act of 2003 - Amends Federal copyright law to: (1) include analog or digital transmissions of a copyrighted work within fair use protections; (2) provide that it is not a copyright infringement for a person who lawfully obtains or receives a transmission of a digital work to reproduce, store, adapt, or access it for archival purposes or to transfer it to a preferred digital media device in order to effect a non-public performance or display; (3) allow the owner of a particular copy of a digital work to sell or otherwise dispose of the work by means of a transmission to a single recipient, provided the owner does not retain his or her copy in a retrievable form and the work is sold or otherwise disposed of in its original format; and (4) permit circumvention of copyright encryption technology if it is necessary to enable a noninfringing use and the copyright owner fails to make publicly available the necessary means for circumvention without additional cost or burden to a person who has lawfully obtained a copy or phonorecord of a work, or lawfully received a transmission of it. | 2,262 | 284 | billsum | en |
Summarize: Jamil Afridi, left, brother of a Pakistani doctor Shakil Afridi, says family and attorneys have been unable to meet with doctor convicted of helping the CIA find Osama bin Laden. (Mohammad Sajjad/AP) Pakistan’s new intelligence chief has postponed his first visit to Washington amid harsh U.S. criticism of the 33-year prison sentence imposed on Shakil Afridi, the Pakistani doctor convicted of treason for aiding the CIA’s hunt for Osama bin Laden. Lt. Gen. Zaheer ul-Islam, appointed in March to head the powerful Inter-Services Intelligence agency, was set to meet this week with his U.S. counterpart, CIA Director David H. Petraeus, a Pakistani official said. But Islam canceled the trip because of “pressing commitments here,” the Pakistani military said in a brief statement Monday. “There is no other reason,” it added. A senior Pakistani official said increased bilateral tensions rooted in the Afridi case and a long-simmering dispute over Pakistan’s refusal to reopen its territory to NATO supply convoys contributed to the postponement. The official, who spoke on the condition of anonymity because of the sensitivity of the matter, said the CIA and the ISI are working on a new date for the meeting. Pakistani doctor Shakil Afridi, who helped the U.S. track down bin Laden, was sentenced to 33 years in prison for conspiring against the state, officials said. (Qazi Rauf/AP) The CIA declined to comment. The ISI detained Afridi, a government surgeon, three weeks after U.S. commandos killed bin Laden. Afridi, 48, ran a fake hepatitis vaccination drive to collect bin Laden’s DNA as a way to verify the al-Qaeda leader’s presence in Abbottabad, the garrison town in Pakistan where he hid for six years until his death. Senior U.S. officials and several members of Congress have expressed outrage at Pakistan’s punishment of Afridi, while lauding him as a hero and patriot. But Pakistani leaders call him a traitor who spied for a foreign power that launched an illegal unilateral operation. For years, U.S. intelligence worked with the ISI to help identify al-Qaeda targets in Pakistan and around the world. That cooperation extended to the CIA’s drone war in Pakistan’s tribal region that has eliminated scores of militants. But the U.S. decision not to inform Pakistan about the impending raid on bin Laden’s compound last May showed the degree to which Washington mistrusted its supposed ally’s spy agency and army. When Islam replaced Lt. Gen. Ahmed Shuja Pasha, who retired after leading the Pakistani spy agency since 2008, some in Washington saw an opening for improving the troubled U.S.-Pakistan alliance against terrorism. The Afridi case is the biggest bilateral problem to occur on Islam’s watch. Members of Congress and top Obama administration officials have called on Pakistan to pardon and release Afridi, and some have implied that Pakistan is an ally of al-Qaeda. On Monday, relatives and supporters of Afridi said at a news conference in the northwestern city of Peshawar that they will seek to overturn his conviction, handed down last week in a tribal court. Under that system, Afridi did not have the right to present evidence or have an attorney. “The United States should help us,” Jamil Afridi, 50, brother of the doctor, said in an interview. Peshawar prison authorities have barred him and the family’s attorneys from communicating with Shakil Afridi, saying a no-visitors policy had been imposed for his safety. The doctor’s wife and three children have not seen him for a year, according to Jamil Afridi. Shakil Afridi has a right to an appeal under rules that govern the tribal areas, but lawyers seeking to represent him said they have been unable to obtain a copy of the court’s verdict, which they need to file an appeal. They also said they have been barred from getting the doctor’s signature, which they need to establish power of attorney so they can represent him. Afridi lived in the semiautonomous Khyber Agency in Pakistan’s northwest, which the government says put him under the jurisdiction of tribal administrative officials who also are empowered to act as a court. Pakistan has made clear that it considers the case an internal judicial matter and has otherwise not responded to U.S. calls for Afridi’s release. Although they initially applauded bin Laden’s death, Pakistan’s leaders have since made it official policy to denounce the Abbottabad raid as a violation of national sovereignty. Pakistan’s politicians and its public also view the CIA drone-fired missile strikes on suspected militants in the tribal belt as an affront to their nation’s sovereignty. Washington has ignored Islamabad’s repeated demands that it end such attacks. The fifth drone strike in less than a week occurred Monday, local officials said, when missiles killed five suspected Islamist extremists in North Waziristan, where many militant groups are based. The relentless attacks have focused on areas near the Afghan border thought to harbor fighters allied with al-Qaeda. Local authorities and tribesmen say that at least 30 fighters have died in the strikes since Wednesday, with many of the victims described as foreign jihadists, including Uzbeks and Arabs. Special correspondent Haq Nawaz Khan in Peshawar contributed to this report. ISLAMABAD (Reuters) - The Pakistani doctor who helped the CIA find Osama bin Laden faced accusations of corruption and other wrongdoing long before he was captured by Pakistani intelligence agents and then jailed for 33 years for treason. Pakistani doctor Shakil Afridi talks with people outside a building at an unknown location in Pakistan in this still image taken from file footage released on May 23, 2012. REUTERS/Geo News via Reuters TV In interviews over the weekend, several current and former Pakistani officials described the doctor, Shakil Afridi, as a hard-drinking womanizer who had faced accusations of sexual assault, harassment and stealing. They said his main obsession was making easy money. According to a 2002 Pakistan health department document seen by Reuters, Afridi was deemed to be corrupt and unreliable and unfit for government service. U.S. officials have hailed Afridi, aged in his 40s, as a hero for helping pinpoint bin Laden’s location in the Pakistani town of Abbottabad where the al Qaeda leader was killed in May last year in a raid by U.S. Navy SEALs. Officially Pakistan has said nothing about Afridi except that the court’s decision to sentence him should be respected. But the fresh accusations about Afridi’s character, coupled with his imprisonment, will almost certainly lead to further strain on already tense bilateral ties. Pakistani officials’ attempts to cast doubt on Afridi’s character will likely be viewed in some quarters as retaliation for his work with the Americans, despite the disclosures in the 2002 Pakistani document. U.S. officials on Monday called the accusations character assassination. In Washington, one senior official said the U.S. government was unaware of any questionable behavior by Afridi. “Available information showed Afridi was a respected member of the Pakistani health care community,” said the senior official. “We are aware of efforts, put in place since Dr. Afridi’s arrest, to denigrate his character.” Another U.S. official said: “It’s nothing short of puzzling that Pakistani officials would disparage someone who helped in the hunt for bin Laden, a terrorist who had Pakistani blood on his hands.” The Afridi family’s lawyer declined to be drawn on the controversy. “I cannot comment on any past allegations against him,” Raza Safi told Reuters. Afridi ran a vaccination campaign in Abbottabad and used cheek swabs to try to gather DNA from bin Laden’s children, said one former Pakistani security official familiar with the case. Accompanied by three health workers, he went to bin Laden’s house and told his wives that a vaccination program was underway in the area, said the former security official. “A woman went in (to the house) and said ‘bring the children out, the doctor is waiting and he will give them the drops’,” the former official said. “That’s when he used the swabs.” It was unclear whether the CIA used the swabs to determine if the children were bin Laden’s. A DNA test can prove close blood relations and U.S. authorities could have matched the samples with profiles it had collected from several of bin Laden’s relatives. INADVERTENTLY CONFIRMED In Washington, another senior U.S. official with knowledge of Afridi’s work for the CIA said the doctor’s vaccination efforts had also enabled him to gather intelligence on bin Laden’s couriers who visited the house. “Dr Afridi was inadvertently able to confirm something we already suspected - that bin Laden’s couriers practiced extraordinary operational security,” the official said. “Was that a key to the raid? No. Was it important? Absolutely.” U.S. Defense Secretary Leon Panetta said on Sunday that Afridi “was not working against Pakistan. He was working against al Qaeda. And I hope that ultimately Pakistan understands that”. “Because what they have done here, I think, you know, does not help in the effort to try to re-establish a relationship between the United States and Pakistan.” U.S. Congressman Dana Rohrabacher introduced legislation in February calling for Afridi to be granted U.S. citizenship and said it was “shameful and unforgivable that our supposed allies” charged him. UNWANTED SCRUTINY Infuriated by the unilateral U.S. raid in a town just a two-hour drive from the capital Islamabad, many in the country see Afridi as a villain who conspired against the state and brought unwanted scrutiny of Pakistan’s attitude to militants. Last week, a tribal court sentenced him to 33 years in prison for working with a foreign intelligence agency. Afridi is being kept in solitary confinement in a prison in the city of Peshawar for fear that he may be targeted by Islamic militants also incarcerated there, said prison sources. Afridi had been working with the CIA for years before the bin Laden raid, providing intelligence on militant groups in Pakistan’s unruly tribal region, said the former Pakistani security official and a former Pakistani intelligence official. They and other officials said he was of questionable character. “Afridi was known to perform surgeries even though his qualification was basic and he was not authorized to conduct surgery,” a senior provincial health official said. “He was accused of conducting surgeries of the eyes, nose, ears, kidneys.” Afridi was also in contact with militant groups and treated Taliban fighters who were wounded in battle with the Pakistani military, said the former security official. The Taliban are described by the state as terrorists, and most Pakistanis strongly oppose their suicide bombing missions, and philosophy. “Keeping in view his extreme lust for money, I am ashamed to even call him a doctor. He is a corrupt, unreliable and low category officer,” said a March 2002 provincial health department report on Afridi’s performance and conduct. The document described Afridi as unreliable, cruel and inhumane and gave him the lowest job performance scores in most categories. It went on to say: “If his overall character as a doctor is taken into account I would recommend and feel that he is not at all fit for government service or any position where money is involved.” CHARACTERLESS Tariq Hayat, formerly the highest government official in the Khyber tribal region, said he knew Afridi when the doctor worked at a hospital there and was a senior medical officer. Hayat said he met him twice to question him over allegations that he had sexually assaulted a nurse at his hospital and had stolen its electrocardiograph machines for his private practice. “I made him stand... I told him you are a characterless person, you have no principles,” said Hayat, adding he had Afridi fired and expelled him from Khyber. “I said ‘you are a thief, doctor’.” A senior health official who said he saw a record of the case said a nurse had complained about sexual harassment to the regional health director. That account was confirmed by a senior police official who investigated Afridi. “A number of nurses had complained about him, that he had behaved inappropriately with them,” said the police official, adding that Afridi was also accused of stealing material sent by international aid agencies and selling it. These accounts could not be independently verified. Afridi’s brother Jamil described the treason charges as baseless and said the doctor was being made a scapegoat. “If my brother had done something wrong, he had a valid U.S. visa. He could have fled the country,” he said, adding that the family had received no offers of help from the U.S. government. He did not respond to questions about the charges of corruption and harassment. “I am in hiding because my life is in danger, all of our lives are in danger,” Jamil Afridi said. “The family is safe for now but the propaganda campaign in the media is putting us in a lot of danger.” Some health workers who knew Afridi described him as a dedicated, polite professional. “He was very nice to all the people in the team and did his job very diligently,” said Naseem Bibi, a nurse. She said she had been with him when the medical team visited bin Laden’s house. “Yes, he was very interested in this house on that day, but I wasn’t sure why,” she said. EASY PREY His reputation hurt by allegations, Afridi was easy prey for the CIA which found him through his connections to Western aid agencies in about 2009, said the former security official. “The man was living beyond his means after he was fired,” said the former security official. “He got married a third time. He maintained a couple of cars.” Afridi, who came to Abbottabad to carry out the vaccination campaign apparently at the CIA’s behest, blundered when he visited the district health officer in the town. He told the officer he was a volunteer who wanted to provide vaccinations in a certain area and he gave the officer his real name, the former security official said. The team moved from house to house conducting vaccinations and leaving chalk marks on the door to show the people inside had been vaccinated, as is customary in Pakistan. “They went in systematically the way a team is supposed to work,” said the official. “No eyebrows were raised.” But after bin Laden was killed, his widows unwittingly helped Pakistani authorities track Afridi. “They said that the only time when somebody from outside visited the house, was this polio vaccination (team),” said the former security official, who believed the only other visitor to the house was bin Laden’s courier, about once a month. Slideshow (4 Images) Afridi was quickly scooped up by security officials. When interrogated, Afridi initially said he had no ties with Americans, said the former security official. “He categorically denied everything to start with,” said the former security official. “But when the Americans started asking for him, then I think the cat was out of the bag.” | Summary: Pakistani officials say Dr. Shakil Afridi was a bad egg who had been accused of sexual assault, harassment, theft, and more long before he was arrested for helping the US find Osama bin Laden. Officials showed Reuters a 2002 document in which Afridi was declared too corrupt for government service. "Keeping in view his extreme lust for money, I am ashamed to even call him a doctor," it reads. Officials say Afridi was known to perform surgeries, even though he wasn't qualified to do so. The US considers Afridi a hero, and is incensed that Pakistan sentenced him to 33 years in prison for treason; Reuters suggests that these new accusations will likely further weaken US-Pakistan relations. "Available information showed Afridi was a respected" doctor, one US official says. "We are aware of efforts... to denigrate his character." The row over Afridi was one of the reasons the new head of Pakistan's ISI postponed a visit to Washington this week, sources tell the Washington Post. Yesterday, Afridi's family held a press conference, at which they urged the US to intervene on his behalf. | 3,462 | 266 | multi_news | en |
Write a title and summarize: The latent EBV nuclear antigen 3C (EBNA3C) is required for transformation of primary human B lymphocytes. Most mature B-cell malignancies originate from malignant transformation of germinal center (GC) B-cells. The GC reaction appears to have a role in malignant transformation, in which a major player of the GC reaction is Bcl6, a key regulator of this process. We now demonstrate that EBNA3C contributes to B-cell transformation by targeted degradation of Bcl6. We show that EBNA3C can physically associate with Bcl6. Notably, EBNA3C expression leads to reduced Bcl6 protein levels in a ubiquitin-proteasome dependent manner. Further, EBNA3C inhibits the transcriptional activity of the Bcl6 promoter through interaction with the cellular protein IRF4. Bcl6 degradation induced by EBNA3C rescued the functions of the Bcl6-targeted downstream regulatory proteins Bcl2 and CCND1, which resulted in increased proliferation and G1-S transition. These data provide new insights into the function of EBNA3C in B-cell transformation during GC reaction, and raises the possibility of developing new targeted therapies against EBV-associated cancers. B-cell development through the germinal center (GC) is controlled strictly by sequential activation or repression of crucial transcription factors, executing the pre- and post-GC B-cell differentiation [1]. The deregulation of induced GC reactions during B-cell development is associated with malignant transformation giving rise to different types of lymphoma and leukemia [2]. Most mature B-cell malignancies, including diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL) and Burkitt’s lymphoma (BL) are derived from malignant transformation of GC B-cells [2,3]. Furthermore, DLBCL is the most common subtype of non-Hodgkin’s lymphoma (NHL), accounting for approximately 40% of all cases [4]. DLBCL is considered a heterogeneous group of tumors, with some specific clinicopathological variants of DLBCLs being associated with the presence of EBV [5,6]. A major regulator of the GC reaction is represented by B-cell lymphoma 6 (Bcl6), a sequence specific transcriptional repressor [7–9]. Knock-out of Bcl6 in vivo results in lack of GC formation and the maturation of high-affinity antibodies [10,11]. Interestingly, deregulation of Bcl6 expression can be found in BL, FL and DLBCL [12,13]. In addition, Bcl6 is the most frequent oncogene involved in roughly 40% of the cases of DLBCLs, and its locus is frequently rearranged due to chromosomal translocations in DLBCL [14,15]. As a key transcriptional repressor in normal B-cell differentiation, Bcl6 was shown to repress NF-κB and the positive regulatory domain I element (PRDM1) also known as Blimp-1 in DLBCLs [16–18]. Also, Bcl6 is now been investigated as a potential therapeutic target for the treatment of tumors with rationally designed specific Bcl6 inhibitors [19–21]. EBV is a lymphotropic virus that is linked to many kinds of B-cell malignancies, including BL, FL and DLBCL [22,23]. EBV infection transforms primary human B-cells into continuously growing lymphoblastoid cells (LCLs) and different latent types were established in EBV-infected cells [23,24]. During latency III or the growth program, EBV expresses the full complement of oncogenic latent proteins, including EBV nuclear antigens EBNA1, EBNA2, EBNA3A, EBNA3B, EBNA3C and EBNA-LP, as well as latent membrane proteins LMP1, LMP2A and LMP2B in addition to numerous RNAs and miRNAs [25]. Genetic studies using recombinant virus strategies demonstrated that EBNA1, EBNA2, EBNA3A, EBNA3C, EBNA-LP and LMP1 are essential or very important for EBV-mediated transformation of primary B-cells in vitro [26–28]. Specifically, EBNA3C has the ability to function as a transcriptional activator and repressor, and can regulate the transcription of both cellular and viral genes [29,30]. A number of earlier studies have shown that EBNA3C interacts with a wide range of transcription factors and modulators, such as c-Myc [31], IRF4 [32], CtBP [33], p53 [34], E2F1 [35] and E2F6 [36], which leads to dysregulation of their associated cellular functions. Previous studies have indicated that expression of EBV latent proteins were associated with Bcl6 expression [37–39]. For example, in some B-cell lymphomas, Bcl6 expression was inversely correlated with LMP1 expression, and some data suggested that LMP1 can cause downregulation of Bcl6 [6,37]. However, the link between LMP1 and Bcl6 was not fully explained as Bcl6 expression was also inhibited in LMP1-negative cells [38]. Similar studies have shown that LMP1 through heterologous expression was unable to suppress expression of Bcl6 in DLBCLs [39]. In addition, EBNA2 can interfere with the germinal center phenotype by downregulating Bcl6 in Non-Hodgkin' s Lymphoma cells [39]. Furthermore, EBV encoded microRNAs can repress Bcl6 expression in DLBCL [38]. However, the mechanism by which Bcl6 is down-regulated in EBV-infected cells is still not fully understood. Our goal is to determine the role of EBNA3C in regulating expression of Bcl6 oncoprotein in B-cells, and further uncover novel molecular mechanisms by which EBNA3C-mediated regulation of cellular functions can lead to B-cell transformation. To determine the expression levels of Bcl6 during EBV infection of primary B-cells, 10 million human peripheral blood mononuclear cells (PBMCs) from two donors, respectively, were infected with wild-type BAC-GFP-EBV or EBNA3C-deleted (ΔE3C) BAC-GFP-EBV. The infected cells were harvested at different time points (0,2, 4,7 and 15 days post-infection), then mRNA was extracted and Real-time PCR was performed for Bcl6 detection. The results showed that for both donors Bcl6 expression was down-regulated and expressed at a relatively low level after wild-type EBV infection. However, its expression was consistently enhanced with the EBNA3C-deleted EBV infection as much as 20–35 fold over wild-type infection (Fig 1A and 1B). This suggested that EBNA3C can play a key role in Bcl6 expression during EBV infection. To determine the effect of EBNA3C on Bcl6 expression in Burkitt’s lymphoma cells, western blot analysis was also performed in EBV-negative BL41 and Akata cells, as well as the corresponding EBV-positive BL41/B95. 8, Akata-EBV cells. We found a significant downregulation of Bcl6 expression in the presence of EBV-infected BL41 and Akata cell lines of approximately 2–4 fold (Fig 1C). To further investigate whether the differential expression was due to the presence of EBNA3C, EBV-negative Ramos and BJAB cells; EBNA3C expressing stable BJAB cells (BJAB7 and BJAB10); and EBV-transformed lymphoblastoid cell lines (LCL1 and LCL2), were analyzed by western blot. Similarly, Bcl6 protein expression was down-regulated close to 50% in the presence of EBNA3C in Burkitt’s lymphoma cells and were dramatically suppressed in EBV-transformed LCLs (Fig 1D). These results strongly suggested that EBNA3C contributes to inhibition of Bcl6 expression. To further examine if Bcl6 expression was regulated by EBNA3C specifically, HEK293T and BJAB cells were transfected with a dose-dependent increase of EBNA3C in addition to heterologous expression of Bcl6. The western blot results demonstrated that EBNA3C expression led to a strong reduction in Bcl6 protein expression in human cells, including B-cell lines of about 4–10 fold (Fig 1E and 1F). To further explore the role of EBNA3C in modulating Bcl6 expression levels in EBV transformed LCLs, EBNA3C knocked-down LCL1 cell line was generated with specific EBNA3C short hairpin RNA (sh-E3C) [40]. Compared to the control LCL1 (sh-Ctrl), Bcl6 protein expression was significantly increased by at least 2-fold in sh-E3C LCL1 cells (Fig 1G). The results provide additional supporting evidence demonstrating that down-regulation of Bcl6 expression can be specifically linked to EBNA3C. Next, we examined whether EBNA3C could interact directly with Bcl6. Two experiments using Co-Immunoprecipitation (Co-IP) assays were performed in different cell types. First, HEK293T cells were transfected with Myc-tagged EBNA3C and HA-tagged Bcl6. The Co-IP results showed that EBNA3C associated in a complex with Bcl6 (Fig 2A). Similarly, an experiment using Saos-2 cells also showed the formation of a complex of EBNA3C and Bcl6 in these cells (Fig 2B). Second, to further determine the interaction in B-cell lines, BJAB, EBNA3C stably expressed BJAB cells (BJAB7 and BJAB10), and EBV-transformed cells (LCL1 and LCL2) were used in our Co-IP experiment. Western blot analysis also validated the above results demonstrating that endogenous EBNA3C can physically associate with Bcl6 in the background of B-cells and more importantly in EBV-transformed lymphoblastoid cell lines (Fig 2C and 2D). To determine the functional binding domain of EBNA3C that specifically interacts with Bcl6, Myc-tagged full length and truncated regions of EBNA3C (1-365aa, 366-620aa, 621-992aa) were co-transfected into HEK293T cells with HA-tagged Bcl6. The targeted protein was immunoprecipitated with anti-Myc or anti-HA antibody, respectively. The results demonstrated that Bcl6 was associated with EBNA3C (366-620aa) along with the full-length EBNA3C protein (1-992aa) (Fig 2E and 2F). Little or no detectable co-immunoprecipitation was observed with the control vector indicating the specificity of the complex between EBNA3C and Bcl6. These results showed that EBNA3C amino acid residues 366-620aa which includes the acidic domain were responsible for the interaction of EBNA3C and Bcl6 protein (Fig 2G). Previous studies have shown that EBNA3C binds to Bcl6 specifically in human cells, so it is expected that these two proteins would be localized within the same cellular compartments. To determine the co-localization of EBNA3C and Bcl6, HEK293T cells were transfected with constructs expressing Myc-tagged EBNA3C and HA-tagged Bcl6, and the cellular localization of the expressed proteins was studied using immunofluorescence microscopy. In cells transfected independently with Myc-EBNA3C or HA-Bcl6 alone, both were found to be primarily expressed in the nucleus (Fig 3A). In cells co-transfected with Myc-EBNA3C and HA-Bcl6, the merged yellow fluorescence demonstrated that EBNA3C co-localized with Bcl6 in human cells (Fig 3A). Similar results were also observed in Saos-2 cells (Fig 3B). To further determine the co-localization of EBNA3C and Bcl6 proteins in more relevant B-cells, immunofluorescence assays were performed using antibodies specific to EBNA3C and Bcl6 proteins in order to examine the endogenous expression in different B-cell lines. The results further confirmed that EBNA3C co-localized with Bcl6 in nuclear compartments of EBV-transformed LCLs (Fig 3C). This was consistent with the results of the above studies which demonstrated that EBNA3C associated with Bcl6 in nuclear complexes as seen in the Co-IP experiments in human cells. To explore the potential mechanism of EBNA3C-mediated down-regulation of Bcl6, a stability assay was performed to determine whether EBNA3C regulated Bcl6 expression at the protein level. HEK293T cells were transfected with HA-tagged Bcl6 and Myc-tagged EBNA3C or Myc-tagged empty vector. Twenty-four hours post-transfection, cells were incubated with protein synthesis inhibitor cycloheximide (CHX) and monitored for Bcl6 protein levels at 0,4, 8,12 hours by western blot analysis. As expected, the results showed that the stability of Bcl6 protein was significantly decreased by greater than 50% in the presence of EBNA3C by 12 hours, while the Bcl6 protein level was more stable in the absence of EBNA3C (compare right and left panels, Fig 4A). To further support these results, BJAB (EBNA3C negative B-cells), and BJAB7 (BJAB stably expressing EBNA3C cells) were treated with CHX for 0,2, 4, and 6 hours. The following western blot analysis also demonstrated that there was a dramatic reduction in the stability of Bcl6 protein which was directly associated with EBNA3C expression as seen by the significant change in the Bcl6 levels by 2 hours post cycloheximide treatment and greater than 50% by 6 hours (compare right and left panels, Fig 4B). Bcl6 expression is strictly regulated during GC reaction, and its degradation through the ubiquitin-proteasome pathway is crucial for B-cell development or lymphomagenesis in temporal function. Earlier studies showed that Bcl6 could be degraded by the ubiquitin-mediated proteasome [41,42]. Therefore, it is expected that Bcl6 degradation is likely mediated by EBNA3C utilizing a similar pathway as EBNA3C has been shown to recruit E3 ligases for targeted degradation of cellular substrates [43,44]. To determine whether this is the case, HA-Bcl6 was transfected along with Myc-EBNA3C or control vector. Twenty four hours post-transfection, the cells were treated with the proteasome inhibitor MG132 for 12 hours or vehicle control. The following western blot analysis showed that EBNA3C promoted the degradation of Bcl6 protein, which was similar to the above results. However, Bcl6 protein expression was increased after MG132 incubation, even in the presence of EBNA3C (Fig 4C). These results demonstrated that the stability of the Bcl6 protein is regulated by EBNA3C via the ubiquitin-proteasome pathway. To further support our hypothesis, ubiquitination assays were performed with different expression plasmids for Myc-E3C, HA-Ub and HA-Bcl6, and incubated for 24 hours followed by MG132 treatment for another 12 hours. This was followed by immunoprecipitation and western blot analysis. The results demonstrated enhanced ubiquitination of Bcl6 when EBNA3C was expressed, when compared with control vector or HA-Ub alone (Fig 4D). This strongly indicated that Bcl6 is likely degraded by expression of EBNA3C through the ubiquitin-proteasome dependent pathway. Bcl6 gene expression is tightly regulated during mature B-cell development [2,45,46]. Our above studies showed that Bcl6 mRNA expression was down-regulated after EBV infection, and that this was associated with EBNA3C expression. To further define how EBNA3C can regulate Bcl6 expression at the mRNA level, different B-cell lines (BJAB, BJAB7, BJAB10, LCL1 and LCL2) were used to monitor endogenous Bcl6 mRNA expression. Bcl6 mRNA expression was significantly greater (>20 fold) in BJAB cells compared to EBNA3C stably expressed BJAB7 and BJAB10 cell, and also EBV-transformed LCL1 and LCL2 cells (Fig 5A). To verify that the inhibition was related to the presence of EBNA3C, EBNA3C stably knocked-down LCL1 (sh-E3C) and the control LCL1 (sh-Ctrl) were used to detect Bcl6 mRNA expression. The results showed that Bcl6 mRNA expression was upregulated significantly after the knockdown of EBNA3C (Fig 5B). In addition, BJAB10 cells were then transfected with specific EBNA3C short hairpin RNA (sh-E3C) to knock down EBNA3C expression. Expectedly, Bcl6 mRNA expression was increased in the EBNA3C knockdown cell lines (Fig 5C). These findings undoubtedly provide new evidence that EBNA3C can inhibit Bcl6 mRNA expression. Bcl6 promoter transcriptional activity could not only be controlled by Bcl6 through binding to the upstream regulatory region of its gene [15], but is also inhibited directly by the transcription factor IRF4 via binding to multiple sites within its promoter [47]. To test whether EBNA3C-mediated Bcl6 mRNA down-regulation was related to its transcriptional activity at the Bcl6 promoter, a dual-luciferase reporter system was implemented. The reporter construct containing a wild-type Bcl6 promoter (pLA/B9) and a dose-dependent increase of Myc-EBNA3C were transfected into cells. Meanwhile, the thymidine kinase promoter-Renilla luciferase reporter plasmid (pRL-TK) was additionally transfected and used as a control for transfection efficiency. The luciferase assay results clearly revealed that the Bcl6 promoter activity was inhibited by EBNA3C in a dose-dependent manner (Fig 5D). Previous experiments showed that EBNA3C did not bind with DNA directly and functions through binding of other cellular transcription proteins to regulate gene expression [48]. Therefore, other transcription proteins mediate the inhibition of viral and cellular genes. EBNA3C interacted with p53, attenuated its function and mediated its degradation [34,44,49]. Furthermore, p53 can activate Bcl6 transcription [50]. It suggests that the transcription activity of the Bcl6 promoter may be inhibited by EBNA3C-induced p53 degradation. However, our results using MEF (p53-/-) cells showed that the regulation of Bcl6 promoter by EBNA3C was independent of the function of p53 protein (S1 Fig). Among several other transcriptional proteins that inhibited Bcl6 promoter, we found that IRF4, a DNA-binding protein, was an important transcription factor for regulating Bcl6 promoter activity [47]. Interestingly, EBNA3C also interacted with IRF4 and contributed to stabilization of IRF4 [32]. One study showed that a high level of IRF4 was expressed in LMP1-KO EBV-induced lymphoma [51]. Thus, we further assessed the possible function of EBNA3C on IRF4-mediated Bcl6 promoter activity. To specifically test the Bcl6 promoter activity, the wild-type and DNA binding domain (DBD) -deleted IRF4 plasmids were used (Fig 5E). The results showed that EBNA3C enhanced the IRF4-mediated inhibition of the Bcl6 promoter activity, and the effect was dependent on the DNA binding domain of IRF4 as the promoter repression was rescued when EBNA3C and IRF4-ΔDBD were co-expressed (Fig 5E). It also suggested that IRF4 is one of the major transcription factors that mediate EBNA3C-regulated inhibition of Bcl6 promoter activity. To examine the effect of EBNA3C on Bcl6-mediated cell proliferation, Saos-2 cells were transfected with expression constructs of EBNA3C and Bcl6, and selected with G418 for two weeks to monitor colony formation. We observed a significant increase in colony numbers when EBNA3C and Bcl6 were co-transfected in comparison to those transfected with only EBNA3C or Bcl6 (Fig 6A). We further extended these studies by performing cell proliferation assays as determined by cell counting for 10 days in Saos-2 cells (Fig 6B). A similar experiment was also repeated in HEK293 cells (Fig 6C). These results demonstrated that expression of EBNA3C and Bcl6 results in a strong induction in cell proliferation. The anti-apoptotic proto-oncogene Bcl2 protein is a critical regulator protein and its expression is inhibited by Bcl6 in GC B-cells [52,53]. Therefore, it was reasonable to believe that EBNA3C-mediated Bcl6 down-regulation will lead to up-regulation of Bcl2 expression. Therefore, its anti-apoptotic function will be activated and leads to promotion of cell proliferation. To determine the expression of Bcl2 in B-cells, LCL1 was treated with a Bcl6-specific inhibitor (79–6) to suppress Bcl6 activity [20]. The Bcl6 inhibitor disrupts Bcl6 transcription activity by binding to its BTB/POZ domain [20]. The following western blot results showed that Bcl2 expression was up-regulated after Bcl6 inhibitor incubation in B-cells by approximately 2-fold (Fig 6D). To further support the results, Bcl2 mRNA expression was determined in the stable EBNA3C or Bcl6 knock-down LCL1 cells to verify that EBNA3C promoted Bcl2 up-regulation through Bcl6 down-regulation in LCLs (S2 Fig). This suggests that EBNA3C-mediated Bcl6 inhibition can contribute to cell proliferation through the Bcl2-associated signaling pathway. The soft agar assay for colony formation measures anchorage-independent in vitro transformation. The oncogene Bcl6 can confer anchorage-independent growth to immortalized cells [54]. To investigate the effects of EBNA3C on Bcl6-related transforming activity, the stable Bcl6 knock-down BJAB and LCL1 cells were generated with lentivirus transduction and puromycin selection (Fig 7A–7C). Soft agar assays were performed using the Bcl6 knock-down BJAB and LCL1 cells. The down-regulation of Bcl6 inhibited the ability of colony formation in BJAB cells (Fig 7D), but oppositely, the ability was enhanced in EBV-transformed LCLs (Fig 7E). The results indicate that EBV promotes transformation and anchorage-independent growth through the inhibition of Bcl6 expression. Our previous study showed that EBNA3C could only stabilize Cyclin D1 (CCND1) protein, but not promote its transcription activity [40]. This suggests that other cellular factors may regulate CCND1 expression. Interestingly, CCND1 is induced by Bcl6 in human B-cells [55]. To further investigate the function of Bcl6 in cell cycle, we analyzed CCND1 mRNA expression in stable B-cells. The results show that CCND1 expression is also suppressed when Bcl6 is knocked-down in EBV-negative BJAB cells. However, its expression is upregulated in stable Bcl6 knock-down EBV-transformed LCL1 cells (Fig 8A and 8B). These results suggest that Bcl6 plays a critical role in controlling CCND1 mRNA expression in a B-cell background. The following cell cycle experiments also demonstrated that the upregulation of CCND1 through Bcl6 inhibition facilitates G1-S transition in EBV-transformed LCL1 cells, but not in the EBV-negative BJAB cells (Fig 8C and 8D). Similar results were observed with other sh-Bcl6 clones. Bcl6 is a nuclear phosphoprotein of the BTB/POZ/Zinc Finger (ZF) protein family, and functions as a transcription repressor to repress target genes by binding to specific DNA sequences and recruiting corepressors [8,56], including SMRT, MTA3, N-CoR and HDAC [57–60]. Bcl6 is indispensable for GC formation and somatic hypermutation (SHM) during B-cell development, thus chromosomal translocations and mutations of Bcl6 regulatory region lead to the deregulation of Bcl6 expression in about 40% DLBCL and 5–10% FL [46]. Although Bcl6 expression is associated with EBV latent antigen EBNA2 and LMP1, the reported conflicting results did not provide a reasonable explanation or a detailed mechanism on EBV-mediated Bcl6 degradation in B-cell lymphoma [37–39]. A recent study indicated that EBNA3C had no effects on Bcl6 expression, but a previous paper also showed that Bcl6 expression can be increased more than 10-fold in EBNA3C-deleted EBV infection [61,62]. Here, our data clearly show that Bcl6 expression can be down-regulated by EBNA3C specifically at transcriptional and post-transcriptional levels (Fig 9). This is different from the well-known Bcl6 translocations or mutations identified on the human genome associated with oncogenesis. First of all, our results demonstrated that EBNA3C was specifically associated with Bcl6, and mediated Bcl6 protein degradation through the ubiquitin-proteasome dependent pathway. A previous study showed that Bcl6 protein can be targeted for degradation by cellular factor FBXO11 in DLBCL [63]. However, the role of EBNA3C on FBXO11-related Bcl6 stability is still unclear. It is possible that FBXO11 is the E3 ligase recruited by EBNA3C for Bcl6 degradation. In addition, the acetylation of Bcl6 within the PEST domain inactivates its function of recruiting co-repressors [54], and the activation of MAPK signaling pathway induces phosphorylation of Bcl6 followed by degradation through the ubiquitin-proteasome pathway [64]. Further studies are warranted to determine whether EBNA3C-mediated Bcl6 degradation is related to Bcl6 acetylation and phosphorylation. BCL6 activity is also dysregulated by translocation or mutation in a remarkably high proportion of DLBCL and FL [65]. The chromosomal translocations of Bcl6 regulatory region referred to as promoter substitution, and frequent mutations of the 5’ noncoding region of Bcl6 result in its deregulated expression, suggesting a key role for Bcl6 in pathogenesis of B-cell lymphoma [12,13,66,67]. However, our results indicate that Bcl6 mRNA expression is down-regulated through EBNA3C-mediated inhibition of the transcription activity of Bcl6 promoter by recruiting another cellular factor IRF4. This is consistent with a previous study showing that the CD40 receptor signaling pathway leads to NF-κB-mediated IRF4 activation, and furthermore Bcl6 downregulation [47]. Meanwhile, we also showed that EBNA3C could interact with IRF4 and was critical for IRF4 stabilization [32]. This suggests that EBNA3C may mimic the activities of the CD40 ligand to induce NF-κB-IRF4 signaling pathway or enhance the stability of IRF4 protein directly to repress the transcription activity of the Bcl6 promoter. Moreover, EBNA3C was associated with IRF8 and mediated its destabilization and degradation [32]. Interestingly, IRF8 is the only transcriptional activator of Bcl6 to upregulate its mRNA expression in GC reaction [68]. Therefore, it is conceivable that EBNA3C may downregulate Bcl6 expression by activating CD40 signaling pathway as well as regulating IRF4/IRF8 stability. The importance of Bcl6 function in GC B-cells is reflected in the multiple functional pathways it can regulate in the cell. To date, more than one thousand genes are found to be targeted by Bcl6 through binding on their promoters and further modulating the downstream signaling pathways during GC development, involved in cell apoptosis, cell cycle and cell differentiation [69,70]. Among the targeted proteins, Bcl2 is a critical anti-apoptosis protein and the direct target of Bcl6 that can interact with Miz1 and bind to Bcl2 promoter to inhibit Miz1-induced Bcl2 transcription activity in GC B-cells [52,53]. The dysregulation of Bcl6-mediated Bcl2 expression is often found in DLBCL and FL [2]. Our results show that Bcl2, a Bcl6 target protein, is regulated by EBNA3C and is increased in LCLs treated with a Bcl6 inhibitor. Thus EBNA3C can induce cell proliferation by degrading and inhibiting the expression of Bcl6 and so releasing the suppression of Bcl2, therefore activating the anti-apoptosis pathway for tumorigenesis. Moreover, CCND1, a direct target of Bcl6 in human B-cells, is de-repressed to promote G1-S transition in EBV-transformed LCLs. Whether other cyclin proteins are also under the control of Bcl6 is still unknown. Interestingly, several studies have shown that CCND2 is another target of Bcl6, but its expression is negatively correlated [71–75]. Activation-induced cytidine deaminase (AID) which is responsible for somatic hypermutation and class-switch recombination is also required in GC-derived lymphomas, and its expression is upregulated by EBNA3C in EBV-infected cells [61,76]. Bcl6 could promote AID expression by inhibiting miR-155 and mir-361, so how EBNA3C regulates AID expression without the help of Bcl6 needs to be further explored [2]. A recent study concluded that Bcl6 targeted genes in T follicular helper (Tfh) cells through analysis of its genome-wide occupancy and transcriptional regulatory networks [77]. The current development of Bcl6 small-molecular inhibitor indicates a huge potential for Bcl6 as a therapeutic target to treat human lymphomas [19]. However, the Bcl6-mediated regulatory networks are still unknown in EBV-transformed LCLs. Next, xenografts of LCLs in Bcl6 knock-out mice will further reveal the biological function of Bcl6 in EBV-related lymphomagenesis. However, a more efficient in vivo model will be necessary to uncover the crucial functions of EBNA3C or other latent antigens in GC reaction. In summary, the inhibition of Bcl6 expression by the essential EBV antigen EBNA3C may provide a novel insight into the current understanding of EBV contribution on lymphomagenesis by blocking GC reaction. Importantly, a number of EBV latent proteins are expressed in EBV infected cells, but how these latent proteins cooperate with each other to regulate B-cell development, or lead to B-cell lymphoma still needs further investigation. Nevertheless, our observations have implications for emerging strategies targeted at the EBV-associated cancers. The University of Pennsylvania Immunology Core (HIC) provided us human peripheral blood mononuclear cells (PBMC) from different unidentified and healthy donors with written, informed consent. All the procedures were approved by the Institutional Review Board (IRB) and conducted according to the declarations of Helsinki protocols [36,78]. Myc-tagged full-length EBNA3C or its truncations such as 1-365aa, 366-620aa, 621-992aa, and Flag-tagged IRF4 plasmids have been described previously [32]. Myc-tagged constructs expressing full length or DNA binding domain (DBD) mutant IRF4, and HA-tagged full length Bcl6, wild-type Bcl6 promoter plasmids [9,54] were kindly provided by Dr. Riccardo Dalla-Favera (Columbia University, New York, USA). HEK293 or HEK293T (human embryonic kidney cell line), Saos-2 (human osteosarcoma cell line), EBV-negative or -positive cells have been described earlier in detail [32,36]. MEF (mouse embryonic fibroblast cell line) was a gift from Xiaolu Yang (University of Pennsylvania) [79]. HEK293, HEK293T, Saos-2, MEF (p53-/-) and MEF (p53+/+) cells were grown in Dulbecco' s modified Eagle' s medium (DMEM), while B-cell lines were maintained in RPMI 1640 media. All the above-mentioned cells were incubated at 37°C in a humidified 5% CO2 environment. The Bcl6 inhibitor (79–6) was purchased from EMD Millipore (Billerica, MA, USA). Bcl6 antibody (N-3) and Bcl2 antibody (C-2) were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA). Bcl6 antibody (ab19011) were purchased from Abcam (Cambridge, UK). Antibodies for IRF4, Ub, GAPDH have been described earlier [32]. Flag antibody (M2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Other antibodies to mouse anti-Myc (9E10), anti-HA (12CA5), anti-EBNA3C (A10) were prepared from hybridoma cultures and mentioned previously [80]. 10 million transfected cells or 50 million B-cells were harvested, washed with ice-cold 1×PBS twice, lysed in 400μl ice-cold RIPA buffer [1% Nonidet P-40 (NP-40), 10 mM Tris (pH8. 0), 2 mM EDTA, 150 mM NaCl, supplement with protease inhibitors (1 mM phenylmethylsulphonyl fluoride (PMSF), 1 μg/ml each aprotinin, pepstain and leupeptin]. Lysates were precleared with normal control serum plus 30 μl of a 2: 1 mixture of Protein-A/G Sepharose beads (GE Healthcare Biosciences, Pittsburgh, PA) for 1 h at 4°C. Approximately 5% of the lysate was saved as input. About 1 μg of specific antibody was used to capture the protein of interest by overnight rotation at 4°C. Input and IP samples were boiled in laemmli buffer, resolved on SDS-PAGE gel and transferred to a 0. 45 μm nitrocellulose membrane. The membrane was blocked in 1×TBS-Tween with 5% w/v non-fat dry milk probed with appropriate primary antibody, subsequently incubated with corresponding secondary antibody, and visualized on a Licor Odyssey imager (LiCor Inc., Lincoln, NE). Image analysis and quantification measurements were performed using Image Quant application software (LiCor Inc., Lincoln, NE). The relative density (RD) of indicated proteins were shown. HEK293T or Saos-2 cells plated on coverslips were transfected with expression plasmids or not as indicated. Forty eight hours post-transfection, cells were fixed by 4% paraformaldehyde (PFA) including 0. 1% Triton X-100 for 15–20 mins at room temperature [81]. B-cells were air-dried and fixed similar to above. The fixed cells were washed with 1×PBS for three times, and 5% Bovine serum albumin (BSA) was used for blocking. EBNA3C and Bcl6 were detected by mouse anti-EBNA3C (A10) and rabbit anti-Bcl6 antibody, respectively. The slides were examined using an Olympus Fluoview 300 confocal microscope, and Images were analyzed by Fluoview software (Olympus Inc., Melville, NY). HEK293T cells were co-transfected with pLA/B9 plasmid (Bcl6 promoter, a gift from Dr. Riccardo Dalla-Favera) [47], pRL-TK (Promega, Madison, WI, USA), Myc-tagged EBNA3C, and control or Myc-tagged IRF4/IRF4-ΔDBD plasmids. Forty eight hours post-transfection, cells were harvested and the dual-luciferase reporter assay was performed according to the manufacture’s protocols (Promega, Madison, WI, USA). At the same time, the supernatant was collected and prepared for detection by western blot. Cells were collected and washed with ice-cold 1×PBS prior to RNA isolation. Then total RNA extraction was performed using Trizol reagent (Invitrogen, Inc., Carlsbad, CA) and treated with Dnase I (Invitrogen, Inc., Carlsbad, CA), then cDNA was prepared with Superscript II reverse transcriptase kit (Invitrogen, Inc., Carlsbad, CA) according to the manufacturer’s protocol. Primers for GAPDH were 5′-TGCACCACCAACTGCTTAG-3′ and 5′-GATGCAGGGATGATGTTC-3′ [40]. Quantitative Real-time PCR analysis was performed by using SYBR green Real-time master mix (MJ Reserch Inc., Waltham, MA). The assays were performed in triplicate. Transfected HEK293T cells were treated with protein synthesis inhibitor cycloheximide (CalBiochem, Gibbstown, NJ) after 24 hours transfection as 40 μg/ml concentration. Cells were harvested after 16 hours incubation and lyse with RIPA buffer, then protein samples were quantitated and used for western blot analysis. Protein band intensities were quantified using Image Quant 3. 0 software. 10 million HEK293 or Saos-2 cells were transfected with control vector, Myc-EBNA3C, Myc-Bcl6 and GFP vector by electroporation and allowed to grow in DMEM supplemented with 1 mg/ml G418 (Sigma-Aldrich, St. Louis, MO, USA). After two weeks selection, GFP fluorescence of every plate was scanned by PhosphorImager (Molecular Dynamics, Piscataway, NJ) and the area of the colonies measured by using Image J software (Adobe Inc., San Jose, CA). Three independent experiments were performed. The two sense strands of Bcl6 shRNA are 5’-tcgagtgctgttgacagtgagcgaGCCTGTTCTATAGCATCTTTAtagtgaagccacagatgtaTAAAGATGCTATAGAACAGGCgtgcctactgcctcggaa–3’ (sh-Bcl6-1), and 5’- tcgagtgctgttgacagtgagcgaCCACAGTGACAAACCCTACAAtagtgaagccacagatgtaTTGTAGGGTTTGTCACTGTGGgtgcctactgcctcggaa–3’ (sh-Bcl6-2), respectively. The upper-cases designate Bcl6 target sequences, while lower cases specify hairpin and enzyme sequences. These sense stranded oligos were annealed with their respective anti-sense stranded oligos, and then cloned into pGIPZ vector with Xho I and Mlu I restriction sites. Besides, a negative control was set using a sh-Ctrl plasmid including control shRNA sequence 5’-TCTCGCTTGGGCGAGAGTAAG–3’ (Dharmacon Research, Chicago, IL). Lentivirus production and transduction of B-cell lines has been described previously with a slight modification [32]. A pool of two shRNAs that targeted different regions of the Bcl6 mRNA were co-transfected to generated shRNA-expressing lentiviruses. The BJAB or LCL1 stable cell lines were generated according to the above-mentioned protocols. Approximately, 5 million BJAB or LCL1 stable cells were collected, fixed with 80% ethanol for 2 hours or overnight at -20°C, then washed with 1×PBS and incubated with PI staining buffer (0. 5 mg/ml propidium iodide in 1×PBS, 50 μg/ml RNase A) for 30 minutes to 2 hours at room temperature. The indicated cells were washed with 1×PBS once, resuspended in 500 μl 1×PBS, and analyzed on FACS Calibur (Becton Dickinson, San Jose, CA, USA) using FlowJo software (TreeStar, San Carlos, CA, USA). The soft agar assays were performed using BJAB or LCL1 cells. Briefly, 1 ml of 0. 5% agar in supplemented RPMI media was poured into 6-well plate and set aside to solidify. 0. 5 ml 0. 3% agar/medium containing 2×105 cells was added to the previously plates as the middle layer. Then cells were covered with a top layer of another 1ml 0. 5% agar/medium. After two weeks, colonies were stained with 0. 005% crystal violet for 1 hour, and scanned using a Licor Odyssey system (LiCor Inc., Lincoln, NE). The number of colonies was counted using ImageJ software. Data represented here are the mean values with standard deviation (SD). The significance of differences in the mean values was calculated by performing 2-tailed student' s t-test. P-value of <0. 05 was considered as statistically significant in all our results (*P < 0. 05; **P < 0. 01; ***P < 0. 001; NS, not significant). Epstein-Barr virus (EBV) genome, strain B95-8-GenBank: V01555. 2; EBNA3C (Human herpesvirus 4) -NCBI Reference Sequence: YP_401671. 1; Bcl6 (Homo sapiens) -NCBI Reference Sequence: NM_001130845. 1; IRF4 (Homo sapiens) -NCBI Reference Sequence: NM_002460. 3; Bcl2 (Homo sapiens) -NCBI Reference Sequence: NM_000633. 2; CCND1 (Homo sapiens) -NCBI Reference Sequence: NM_053056. 2; p53 (Homo sapiens) -NCBI Reference Sequence: NM_000546. 5. | Title: An essential EBV latent antigen 3C binds Bcl6 for targeted degradation and cell proliferation Summary: Epstein-Barr virus (EBV) is the first characterized human tumor virus, which is associated with a broad range of human cancers. One of the latent proteins, EBV encoded nuclear antigen 3C (EBNA3C) plays a critical role in EBV-mediated B-cell transformation. Bcl6 is a master regulator required in mature B-cells during germinal center (GC) reaction. As a transcriptional repressor, Bcl6 can be targeted during malignant transformation and therefore contributes to its function as an oncoprotein during lymphomagenesis. In this study, we demonstrated that EBNA3C interacts with Bcl6 and facilitates its degradation through the ubiquitin-proteasome dependent pathway, and suppresses Bcl6 mRNA expression by inhibiting the transcriptional activity of its promoter. Furthermore, EBNA3C-mediated Bcl6 regulation significantly promotes cell proliferation and cell cycle by targeting Bcl2 and CCND1. Therefore, our findings offer new insights into the functions of EBNA3C during B-cell transformation in GC reaction and B-cell lymphoma development. This increases the possibility of developing new therapies for treating EBV-associated cancers. | 10,137 | 291 | lay_plos | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATIONS Not Applicable FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT Not Applicable INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ON A COMPACT DISK Not Applicable BACKGROUND OF THE INVENTION Many offerings exist by which a soccer players can practice their skills. The devices offered, however, do not solve all problems associated with portability. When the existing goals are used, then the player cannot easily alter the layout should the need arise. If more than one player is practicing, occasionally the goals cannot accommodate the quantity of incoming balls. FIELD OF THE INVENTION The present invention relates to a training device for soccer players for use in connection with training soccer players the basics of ball handling. The training device for soccer players has particular utility in connection with teaching close distance aiming and passing skills, as well as long distance aiming and passing skills to children and others desirous of honing their skills. DESCRIPTION OF THE PRIOR ART The use of training devices for soccer players is known in the prior art. For example, U.S. Pat. No. 6,508,729 to Coltrane et al. discloses a goal shot training system. However, the Coltrane '729 patent does not afford the user portability, and has further drawbacks of requiring the use of a certain type of goal system, which might not be the standard in the users field. U.S. Pat. No. 5,746,669 to Sinsheimer et al. discloses a game and training device for teaching soccer skills that uses a series of hoops and a final goal. However, the Sinsheimer '669 patent does not have the structure of the present invention, and additionally does not allow for the ball to pass through the final goal member. While the above-described devices fulfill their respective and particular objects and requirements, they do not describe a training device for soccer players that provides for the advantages of the present invention; therefore, a need exists for an improved training device for soccer players, particularly one that includes portability, ease of set-up and use, and the ability to be used by one player or multiple players. In this respect, the present invention substantially departs from the conventional concepts and designs of the prior art. SUMMARY OF THE INVENTION In view of the foregoing disadvantages inherent in the known types of training devices for soccer players now present in the prior art, the present invention provides an improved training device for soccer players, and overcomes the above-mentioned disadvantages and drawbacks of the prior art. As such, the general purpose of the present invention, which will be described subsequently in greater detail, is to provide a new and improved training device for soccer players and method which has all the advantages of the prior art mentioned heretofore and many novel features that result in a training device for soccer players which is not anticipated, rendered obvious, suggested, or even implied by the prior art, either alone or in any combination thereof. To attain this, the present invention essentially comprises a plurality of connection means having a main body and three hollow engagement ports. The engagement ports angle away from the main body. There is a frame that is made from a pair of front and rear frame members, a pair of side frame members, and a pair of upper frame members. The frame members all have opposite ends, and the ends are sized to fit into the hollow portion of the engagement ports, much as tent poles fit into the tent frame connectors. The frame is covered by a flexible outer covering that supports a ring member placed within an opening formed through or within the flexible outer covering. The ring is the aiming area for the soccer player who is training and using the training device. The outer covering can be made from mesh or fabric, as desired by the manufacturer and end user. The use of these types of covering materials would aide in the use of the training device, as they are lightweight enough to be easily carried and flexible enough to fold and store while the device is in transit or storage. There has thus been outlined, rather broadly, the more important features of the invention in order that the detailed description thereof that follows may be better understood and in order that the present contribution to the art may be better appreciated. The invention may also include one or two aiming rings as desired. There are, of course, additional features of the invention that will be described hereinafter and which will form the subject matter of the claims attached. The ring or rings would be connected to the fabric to ensure stability and strength. It is therefore an object of the present invention to provide a new and improved training device for soccer players that has all of the advantages of the prior art training devices for soccer players and none of the disadvantages. It is another object of the present invention to provide a new and improved training device for soccer players that may be easily and efficiently manufactured and marketed. An even further object of the present invention is to provide a new and improved training device for soccer players that has a low cost of manufacture with regard to both materials and labor, and which accordingly is then susceptible of low prices of sale to the consuming public, thereby making such training device for soccer players economically available to the buying public. Still another object of the present invention is to provide a new training device for soccer players that provides in the apparatuses and methods of the prior art some of the advantages thereof, while simultaneously overcoming some of the disadvantages normally associated therewith. Even still another object of the present invention is to provide a training device for soccer players for easy portability and set up. This allows the user to train with the device as needed or desired, and not be constrained by the use of existing goals or facilities. Still yet another object of the present invention is to provide a training device for soccer players for compact and efficient storage. This makes it possible to store the device when not in use. Thus has been broadly outlined the more important features of the training device for soccer players so that the detailed description thereof that follows may be better understood and in order that the present contribution to the art may be better appreciated. Numerous objects, features and advantages of the training device for soccer players will be readily apparent to those of ordinary skill in the art upon reading the following detailed description of presently preferred, but nonetheless illustrative, embodiments of the training device for soccer players when taken in conjunction with the accompanying drawings. In this respect, before explaining the current embodiments of the training device for soccer players in detail, it is to be understood that the invention is not limited in its application to the details of construction and arrangements of the components set forth in the following description or illustration. The invention is capable of other embodiments and of being practiced and carried out in various ways. It is also to be understood that the phraseology and terminology employed herein are for purposes of description and should not be regarded as limiting. 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 design of other structures, methods and systems for carrying out the several purposes of the training device for soccer players. It is therefore important 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 together with additional objects of the training device for soccer players, along with various novel features that characterize the invention are particularly pointed out in the claims forming a part of this disclosure. For better understanding of the training device for soccer players, its operating advantages and specific objects attained by its uses, refer to the accompanying drawings and description. BRIEF DESCRIPTION OF THE DRAWINGS The invention will be better understood and objects other than those set forth above will become apparent when consideration is given to the following detailed description thereof. Such description makes reference to the annexed drawings wherein: FIGURES FIG. 1 is a front view of the preferred embodiment of the training device for soccer players constructed in accordance with the principles of the present invention. FIG. 2 is a left perspective view of the training device for soccer players. FIG. 3 is a bottom perspective view of the training device for soccer players. FIG. 4 is a side view of the unfolded frame of the training device for soccer players, with the outer covering removed for clarity, showing the rotation of the frame. FIG. 5 is a side view of the folded frame of the training device for soccer players, with the outer covering removed for clarity, showing the rotation of the frame. REFERENCE NUMERALS connection means 12 main body 14 engagement ports 16 front and rear frame members 18, 20 opposite ends 22, 24 side frame members 26, 28 opposite ends 30, 32 upper frame members 34, 36 opposite ends 38, 40 flexible outer covering 42 ring member 44 opening 46 The same reference numbers refer to the same parts throughout the various figures. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Referring now to the drawings, a preferred embodiment of the training device for soccer players of the present invention is shown and generally designated by the reference numeral 10. In FIG. 1, a new and improved training device for soccer players 10 of the present invention for improving skills is illustrated and will be described. More particularly, the training device for soccer players 10 has a plurality of connection means 12 having a main body 14 and three hollow engagement ports 16. The connection means 12 are used at the corners to engage and support the frame members 18, 20, 26, 28, 34 and 36. The training device for soccer players 10 is shown with the frame assembled and the flexible outer covering 42 placed over the frame. The frame is formed from a pair of front and rear frame members 18 and 20, a pair of side frame members 26 and 28, and a pair of upper frame members 34 and 36. The front and rear frame members 18 and 20 have opposite ends 22 and 24, and the ends 22 and 24 are sized for reception in the engagement ports 16. The side frame members 26 and 28 have opposite ends 30 and 32, and the ends 30 and 32 are sized for reception in the engagement ports 16. The upper frame members 34 and 36 have opposite ends 38 and 40, and the ends 38 and 40 are sized for reception in the engagement ports 16. The ring members 44 placed within an opening 46 in the flexible outer covering 42 can be seen placed in both the side and the top of the flexible outer covering 42. The flexible outer covering 42 is shown as mesh in the Figures, but it is anticipated that any suitable material could be used as desired. FIG. 2 is a left perspective view and shows the present invention as in use, with a soccer ball entering along the dashed line into the body of the training device for soccer players 10 through the side ring member 44. The placement of the ring members 44 in the opening 46 formed in the flexible outer covering 42 is shown more clearly. The plurality of connection means 12 having a main body 14 and three hollow engagement ports 16 are shown with the respective ends 22, 24, 30, 32, 38 and 40 of the frame members 18, 20, 26, 28, 34 and 36 inserted in the engagement ports 16. The training device for soccer players 10 is shown with the frame assembled and the flexible outer covering 42 placed over the frame. The frame is formed from a pair of front and rear frame members 18 and 20, a pair of side frame members 26 and 28, and a pair of upper frame members 34 and 36. The front and rear frame members 18 and 20 have opposite ends 22 and 24, and the ends 22 and 24 are sized for reception in the engagement ports 16. The side frame members 26 and 28 have opposite ends 30 and 32, and the ends 30 and 32 are sized for reception in the engagement ports 16. FIG. 3 is a bottom perspective view showing the present invention as in use, with a soccer ball entering along the dashed line into the body of the training device for soccer players 10. In this Figure, an alternate method for using the training device is shown. The frame is placed with one of the upper frame members, 34 or 36, against the ground. The ball thus enters through what would be previously considered the bottom. This placement of the training device would be most useful for multiple players or long distance practice. The connection means 12, main body 14 and three hollow engagement ports 16 are shown in the corners and with the respective ends 22, 24, 30, 32, 38 and 40 of the frame members 18, 20, 26, 28, 34 and 36 inserted in the engagement ports 16. The flexible outer covering 42 is shown placed over the frame members 18, 20, 26, 28, 34 and 36 and supports ring members 44. The opening 46 through the flexible outer covering 42 is of a size to accommodate the entrance of a soccer ball. FIG. 4 is a side view of the unfolded frame. The rotation of the frame members 18, 20, 26, 28, 34 and 36 within the hollow engagement ports 16 and the selective removal of the ends 22, 24, 30, 32, 38 and 40 from the hollow engagement ports 16 can be seen. To disassemble, the ring members 44 is lifted from the upper frame member 34 and the upper frame member 34 is then lifted from the engagement port 16. The upper frame member 34 is then placed inside the frame. The ring members 44 swivels down against the upper frame member 36. FIG. 5 is a side view of the folded frame showing the frame disassembled as for transport or storage. The rotation of the frame members 18, 20, 26, 28, 34 and 36 within the hollow engagement ports 16 and the selective removal of the ends 22, 24, 30, 32, 38 and 40 from the hollow engagement ports 16 can be seen. The upper frame member 36 is removed from the hollow engagement ports 16, and the upper frame member 36 and ring members 44 are then placed inside the frame. The inventor designed the present invention to aide in his coaching of soccer skills, as he had not found a product available that met all his needs. The device would be used to help players practice aimed shots both at the goal and in passing. The present invention can be used upright on the rectangular base and the player shoots for the circular ring opening, or tipped backwards with the frame positioned vertically, and the player shoots for the larger opening this provides. In the first use the player is closer to the goal and is working on finer control and aiming skills, and in the second use the player is likely farther away and is working on distance aiming skills. In use, it can now be understood that the training device for soccer players provides a useful item for developing and practicing soccer aiming and passing skills. The frame can be quickly assembled, and conversely disassembled for storage. The connection means are a lightweight convenient method for assembling the frame and holding the ends so that the outer covering can be attached. The ring openings are used as the goal for aiming the ball into, and the ball then goes out the back for retrieval and further practice. The portability afforded by the present invention increases the opportunities available to a soccer player to practice. Additionally, the convenience of being able to quickly assemble, and disassemble and store the item adds to its attraction. While a preferred embodiment of the training device for soccer players has been described in detail, it should be apparent that modifications and variations thereto are possible, all of which fall within the true spirit and scope of the invention. With respect to the above description then, it is to be realized that the optimum dimensional relationships for the parts of the invention, to include variations in size, materials, shape, form, function and manner of operation, assembly and use, are deemed readily apparent and obvious to one skilled in the art, and all equivalent relationships to those illustrated in the drawings and described in the specification are intended to be encompassed by the present invention. For example, any suitable sturdy material such as metal or plastic may be used for the connection means. Also, the outer covering may also be made of heavy-duty plastic, cloth, or similar material. And although use for increasing soccer players skills have been described, it should be appreciated that the training device for soccer players herein described is also suitable for training skills in any game or sport where accuracy with propelling a ball using the foot is valued. 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: This is a training device for soccer players that uses a plurality of connectors that have a main body and three hollow engagement ports that are angled away from the main body to provide support for the frame. The ends of the frame members fit into the engagement ports and thus engage the frame and support it for use. There is a flexible outer covering that fits over the frame members and supports the ring members that are placed within an opening in the outer covering. There can be one or two rings as desired. The outer covering does not cover the bottom of the frame, and this area can also be used as a target area. | 3,990 | 128 | big_patent | en |
Summarize: The Constitution prohibits excluding someone from a jury based on sexual orientation, a federal appeals court ruled Tuesday, the latest in a string of decisions granting gays and lesbians more legal protections. A lawyer is allowed to strike someone from a jury for nearly any reason as long as it isn't tied to the person's sex or race. In a series of rulings in the 1980s and 1990s, the Supreme Court said that striking jurors on... In a decision giving sweeping new legal protections to gays and lesbians, a federal appeals court on Tuesday found it as unconstitutional to exclude jurors based on sexual orientation as it is to keep women and minorities off juries. The 9th U.S. Circuit Court of Appeals found that sexual orientation deserves the strongest anti-discrimination protections in civil rights law, siding with gay rights advocates who argued that gays and lesbians are entitled to the same equal treatment in jury trials as they are in the military, voting and marriage. The groundbreaking decision underscored the growing importance of recent rulings in gay marriage battles, relying heavily on the Supreme Court's decision last year striking down a federal ban on same-sex marriage benefits. The 9th Circuit also cited high-court precedents forbidding bias in jury selection based on gender and race. Bumping prospective jurors from a trial based on sexual orientation continues a "deplorable tradition of treating gays and lesbians as undeserving of participation in our nation's most cherished rites and rituals," wrote 9th Circuit Judge Stephen Reinhardt in the three-judge panel's unanimous ruling. These biases "deprive individuals of the opportunity to participate in perfecting democracy and guarding our ideals of justice on account of a characteristic that has nothing to do with their fitness to serve." The decision came in a high-stakes showdown between Abbott Laboratories and SmithKline Beecham, rival pharmaceutical giants that squared off in a 2011 federal antitrust trial in Oakland. The issue arose during jury selection, when SmithKline's legal team suggested Abbott deliberately removed a juror who was gay because the trial involved a claim that Abbott dramatically jacked up the price of a crucial HIV treatment drug. Advertisement Abbott's drug pricing provoked a furor in the gay community. The company denied ill motives in removing the juror, but the case created a major test of whether a pivotal 1986 U.S. Supreme Court decision in Batson v. Kentucky, barring the exclusion of jurors based on race, applies to gays and lesbians. The Abbott case also gave lawyers an opportunity to test the scope of the Supreme Court's ruling last June invalidating the federal Defense of Marriage Act. The 9th Circuit invited the two sides to present arguments on how the marriage ruling applied to the jury issue, and a dozen gay rights organizations jumped into the legal fray to side with SmithKline. Gay rights advocates argue that equal treatment in jury service is akin to seeking the same rights in the military, voting and marriage. Abbott's lawyers argued that Supreme Court precedents do not apply to sexual orientation in jury selection and had urged the 9th Circuit to avoid the issue in the antitrust case. But the 9th Circuit took the issue head-on, perhaps putting the case in a position for eventual Supreme Court review. The panel was decidedly liberal: Reinhardt was joined by other members of the court's liberal wing, Judge Mary Schroeder, a Carter appointee, and Marsha Berzon, a Clinton appointee. Legal experts, however, say the Supreme Court may be reluctant to consider this ruling, which could leave a lasting impact in Western states on other cases involving discrimination against gays and lesbians. Vikram Amar, a UC Davis law professor, said the new legal protection could be particularly important in challenges to bans on gay marriage in other states within the 9th Circuit, such as Nevada, which is defending its law in the same court. California's gay marriage ban already has been invalidated. "Today's ruling will make it exceedingly difficult for states to justify laws that discriminate based on sexual orientation," said David Codell, litigation director of the National Center for Lesbian Rights. Meanwhile, as a result of Tuesday's ruling, the 9th Circuit ordered a new trial in the antitrust feud. A federal jury reached a mixed verdict in the first trial over Abbott and SmithKline's dispute over licensing agreements and pricing for an Abbott drug called Norvir. SmithKline said in a statement it was pleased with the ruling, calling it "well reasoned." An Abbott spokeswoman said the company is reviewing its legal options. The company, which has spun off the division that produces the HIV drug, can ask the 9th Circuit to rehear the case with an 11-judge panel. Howard Mintz covers legal affairs. Contact him at 408-286-0236 or follow him at Twitter.com/hmintz. | Summary: Another big step forward for gay rights: A federal appeals court has ruled that keeping gays and lesbians off juries because of their sexual orientation is just as unconstitutional as excluding women or minorities, reports the San Jose Mercury News. The ruling came in connection with a legal battle between pharmaceutical giants Abbott Laboratories and GlaxoSmithKline, in which GSK argued that Abbott's lawyers deliberately removed a gay juror because the case involved pricing of HIV drugs. "Gays and lesbians have been systematically excluded from the most important institutions of self-governance," one judge wrote for a unanimous three-judge panel. "Strikes exercised on the basis of sexual orientation continue this deplorable tradition of treating gays and lesbians as undeserving of participation in our nation's most cherished rites and rituals." For its decision, the San Francisco court relied partly on last year's landmark Supreme Court ruling that struck down the Defense of Marriage Act, notes the Wall Street Journal. | 1,131 | 228 | multi_news | en |
Summarize: By. Mia De Graaf. PUBLISHED:. 13:22 EST, 7 January 2014. |. UPDATED:. 14:03 EST, 7 January 2014. A police officer, her lover and his wife stole thousands of road crash victims' details from her force computer and sold them to injury lawyers in a conspiracy that could have been worth £1million, a court heard. Former constable Sugra Hanif, 27, allegedly downloaded the personal files of people involved in accidents before pressurising them into claiming compensation so she could claim £800 referral fees from solicitors. Hanif was having an affair with co-accused Raza Khan, also 27, for eight months of the 11-month scheme - but ended it when they were arrested after an anonymous tip-off, prosecutors claim. Accused: Sugra Hanif, 27, (left) allegedly downloaded unique reference numbers from Thames Valley Police computers. Raza Khan, 27, and his wife Paramjeet Kaur, 26, are also accused of conspiring to get up to £1million in solicitor referral fees. The jury at Winchester Crown Court heard the lovers, with Khan's wife, Paramjeet Kaur, 26, set up three companies in April 2011 before selling a total of 2,456 unique reference numbers for £26,400 to various firms. Peter Asteris, prosecuting, said: 'If all of the data stolen had been converted into referral fees, if they had kept going, the value of those referral fees would have been worth over £1 million. 'Every day, tens of thousands of police officers go out there and perform their duties sometimes at not inconsiderable risk to themselves, and they do so as we would expect them to, with duty, responsibility and integrity. 'Integrity, honesty and duty are really what this case is all about. One of these defendants, Sugra Hanif, was a serving police officer with Thames Valley Police. Denial: Khan and Kaur, here leaving Winchester Crown Court, claim they did not know the illegal source of the information. 'The Crown's case against her is that she doesn't have integrity, she didn't fulfil her duties to us, the public, in the way she ought and she abused her position and she abused it with the assistance of the other two defendants in the dock. 'All three of them have been involved in a conspiracy to obtain confidential police information.' He continued: 'She had accessed, I am going to suggest, a staggering 2,456 URNs, different incidents, on the computer and almost all of them had no connection with her duties, no connection with her responsibilities as an investigating officer. 'The Crown says she was abusing that system and stealing 2,500 people's details from that computer system. She had on almost every case no valid reason for doing so whatsoever. 'We say it was nothing short of a deliberate and cynical abuse of the privilege she had been given by having access to that system in the first place.' All three defendants deny the charges. Mr Asteris said that on March 2011, Khan. registered a company called SR Auto Repairs Limited and the following. month Hanif began to steal the police data. And Hanif told police that the S and the R in the company. referred to the initials of her first name and Khan's, he added. Khan denies this. Mr Asteris said that the group went on to set. up a second company in May 2011, registered under Kaur's name, to handle. the growing number of cases. He said a third company was then set up, again under Kaur, who claims ignorance of the illegal source of the data. The jury heard Hanif claims Khan threatened and blackmailed her to steal the data, while Khan insists he did not know the source was illegal. Mr Asteris said: 'That defence is an absolute nonsense and doesn't stand up to any scrutiny. 'It. is more than a coincidence that these two people, who are lovers, set up. a company with the initials of their first two names. 'They became lovers and it seems that relationship has continued on for most of time we are dealing with in this case. The jury heard Hanif, here leaving court with her solicitor, set up three companies with her co-accused to pressurise victims into claiming compensation. 'The Crown says this conspiracy was planned out in advance, particularly by Mr Khan and Miss Hanif. 'Miss Hanif appears to suggest to police in her interview that her involvement in taking this data illegally was that she had been forced to by Mr Khan. 'She says that in fact she was being blackmailed and threatened and he threatened to disclose things to her family and employer.' He said Khan sent her an email which included the lines 'I will never stop loving you' and 'Thanks for the great sex' and 'At least you blew out my candle on my birthday'. He explained that of the 500 calls from Hanif's mobile phone to Khan during the period of the conspiracy, many were just before and after she accessed information on the police computer. The prosecutor added that Hanif would also contact the accident victims and use 'pressure sales' techniques. He also said that £1,600 in cash was found by police at Khan's house. The trial, which is expected to last a month, continues | Summary: Sugra Hanif, 27, 'downloaded 2,456 unique reference numbers from Thames Valley Police computers and sold them to injury lawyers for £800 fee each' She and lover Raza Khan deny obtaining personal details and conspiracy. Khan's wife, Paramjeet Kaur, 26, also accused of conspiracy, denies charge. Winchester Crown Court heard trio used 'pressure techniques' on victims. | 1,256 | 101 | cnn_dailymail | en |
Write a title and summarize: Filariae are tissue-invasive nematodes that cause diseases such as elephantiasis and river blindness. The goal of this study was to characterize the role of histamine during Litomosoides sigmodontis infection of BALB/c mice, a murine model of filariasis. Time course studies demonstrated that while expression of histidine decarboxylase mRNA increases throughout 12 weeks of infection, serum levels of histamine exhibit two peaks—one 30 minutes after primary infection and one 8 weeks later. Interestingly, mice treated with fexofenadine, a histamine receptor 1 inhibitor, demonstrated significantly reduced worm burden in infected mice compared to untreated infected controls. Although fexofenadine-treated mice had decreased antigen-specific IgE levels as well as lower splenocyte IL-5 and IFNγ production, they exhibited a greater than fourfold rise in eosinophil numbers at the tissue site where adult L. sigmodontis worms reside. Fexofenadine-mediated clearance of L. sigmodontis worms was dependent on host eosinophils, as fexofenadine did not decrease worm burdens in eosinophil-deficient dblGATA mice. These findings suggest that histamine release induced by tissue invasive helminths may aid parasite survival by diminishing eosinophilic responses. Further, these results raise the possibility that combining H1 receptor inhibitors with current anthelmintics may improve treatment efficacy for filariae and other tissue-invasive helminths. Filariae are vector-borne tissue-invasive nematodes that infect over 100 million people worldwide and cause the debilitating conditions of river blindness and elephantiasis [1]. A major obstacle to ongoing efforts to control and potentially eradicate these diseases is the limited ability of anti-filarial drugs to kill adult worms, especially when given as single dose treatments. One of the fairly unique aspects of helminth infections, in contrast to infection with most other pathogens, is the induction of histamine release in response to the parasites. Like other helminths, filariae induce the production of antigen-specific IgE, which then sensitizes basophils and mast cells to release histamine in response to parasite antigens. Histamine (2-[4-imidazolyl]ethylamine) is a short-acting biogenic amine that, in addition to having potent acute inflammatory properties, also has numerous immunomodulatory effects on chronic inflammation [2]. Histamine is synthesized by the enzyme histamine decarboxylase (HDC) and is either stored in cytoplasmic granules in basophils and mast cells or is immediately released into the periphery [3]. Histamine release from both basophils and mast cells in response to parasite antigen has been observed in numerous studies of helminth infections [2,4–8]. Although sensitivity to parasite antigens is primarily dependent on parasite-specific IgE [9], several helminths can also induce histamine release in the absence of parasite-specific IgE [10]. In this study we investigated the role histamine plays in the immune response to filariae and the effect antihistamine therapy has on filarial worm burdens. Using the Litomosoides sigmodontis/mouse model we observed that administration of fexofenadine, a histamine receptor 1 antagonist (HR1i), reduces adult worm numbers by over 50%. Additionally, clearance of adult worms in HR1i treated mice was found to be primarily eosinophil dependent, as HR1i administration did not enhance worm clearance in eosinophil-deficient infected mice. All experiments were performed under protocols approved by the Uniformed Services University Institutional Animal Care and Use Committee. Female BALB/c (NCI, Frederick, MD), and BALB/c eosinophil deficient (ΔdblGATA mice, The Jackson Laboratory, Bar Harbor ME), were maintained at the Uniformed Services University with free access to food and water. At study endpoints, all animals were euthanized using carbon dioxide followed by cervical dislocation. Blood was collected by cardiac puncture. For Litomosoides sigmodontis infection, L3-stage larvae (L3s) were obtained from infected jirds (Meriones unguiculatus, TRS labs, Atlanta, GA) by pleural lavage with RPMI 1640. 40 L3s were collected and injected subcutaneously (dorsal neck) into 6–10 week old mice as previously described [11]. For microfilarial counts, 30 μl of blood was taken and mixed with 1 mL ACK lysing buffer (Quality Biological). To count microfilarial numbers, the lysate was pelleted and assessed for counts microscopically. Fexofenadine HCl, an HR1 antagonist (HR1i), was dissolved in the drinking water at a concentration of 0. 25 mg/ml for an average daily dosage of 20mg/kg/day. Cimetidine (Sigma-Aldrich), an HR2 antagonist, was prepared by dissolving in hydrochloric acid (HCl) and mixed with water. The pH was then adjusted to 7. 0 with sodium hydroxide (NaOH). The final concentration of cimetidine was 2. 5 mg/ml in drinking water, for an average daily dosage of 200 mg/kg/day. Drinking water bottles containing antihistamines were changed every other day. Antihistamine activity was confirmed by testing stomach pH at time of euthanasia (for HR1 antagonists) and by local anaphylaxis in response to a direct histamine challenge (for HR2 antagonists). Blood was collected at different time points in heparinized plasma separator microfuge tubes (Starstedt, Nümbrecht, Germany). Samples were centrifuged at 15,000 X g for 1. 5 minutes. Histamine in plasma was detected using a commercially available histamine ELISA assay according to the manufacturer’s instructions (Beckman-Coulter). Adult worms were collected from the pleural cavity of infected animals at 8 weeks post infection. Adult worms were fixed overnight in 4% paraformaldehyde and were washed in 70% ethanol prior to histological processing by Histoserv, Inc (Rockville, MD). In brief, the fixed tissue was dehydrated through graded alcohols, cleared in xylene and infiltrated with paraffin. The processed tissue was then embedded in paraffin and sectioned on a microtome at 5 microns. The slides were then deparaffinized in xylene, hydrated through graded alcohols to water then stained with Carazzi’s hematoxylin. Following a water rinse, they were stained with eosin and dehydrated with graded alcohols. The slides were then cleared using xylene and coverslipped with permount. RNA from whole blood was isolated according to the manufacturer’s instructions (Ambion, Mouse Whole Blood RNA isolation). cDNA synthesis was performed using random primers according to the manufacturer’s instructions (iScript cDNA synthesis kit, BioRad). RT-PCR was performed using a murine histidine decarboxylase (HDC) gene expression assay following manufacturer’s instructions. Samples were analyzed using an Applied Biosystems 7500 Real-Time PCR system and results calculated as fold change relative to an endogenous 18s rRNA control using the 2-ΔΔ CT method. L. sigmodontis worm antigen (LsAg) was prepared as previously described [12]. L3 stage larvae were collected from infected jirds as previously described. For in vitro survival assays, 200 L3s were cultured in 5ml of RPMI 1640 medium supplemented with gentamicin. Cultures were supplemented with 200mM histamine or 1mM Fexofenadine HCl and observed daily for mobility to assess survival. To assess eosinophil numbers at site of adult worm infection, pleural cells were collected by pleural lavage. Red blood cells were lysed using ImmunoLyse kit (Beckman Coulter) and then 2. 0x106cells/mL were permeabilized with BD Permeablization/Wash buffer (BD Biosciences). For analysis, cells were blocked using CD16/CD32 (soluble FcεR III/II receptor, BD Pharmingen) and stained for flow cytometry using anti-SiglecF PE, anti-CD11c APC and anti-CD45 FITC (all from BD Pharmingen). Flow cytometry was performed using a BD LSR II system and analyzed with FACSDiVa 6. 1 software (BD Biosciences). Antibodies for all flow cytometry experiments were titrated prior to use. During analysis, cut-offs for CD45 positivity and Siglec F positivity were determined using the fluorescence minus one approach. To assess levels of eosinophil peroxidase at the site of adult worm infection, pleural fluid was collected by pleural lavage using 1 mL of sterile RMPI. EPO in lavage fluid was assessed by ELISA according to manufacturers instructions (US Biological Life Sciences). Statistical analysis was performed using GraphPad Prism software (GraphPad Software, San Diego, Ca). To determine differences between multiple groups, analysis was performed using Kruskal-Wallis test followed by Dunn multiple comparisons. To determine differences between two un-paired groups, Mann-Whitney analysis was performed. A p value of <0. 05 was considered significant. To determine if infection with L. sigmodontis results in detectable histamine release, BALB/c mice were infected with 40 L3 stage larvae by subcutaneous injection and circulating histamine levels assessed at 30 minutes, 1,4, 8 and 12 weeks by competitive ELISA. In this model L3 larvae migrate to the pleural space where they mature to adult worms, start releasing blood-dwelling microfilariae by 7 weeks, and survive for 12–20 weeks. There was a significant peak of circulating histamine observed 30 minutes after injection of L3s and a second, higher peak correlating with the production of microfilariae at 8 weeks of infection (Fig 1A, p < 0. 01 for both timepoints when compared to age-matched uninfected controls). Of note, histamine was not detected in the blood of mice 30 minutes after injection of vehicle (sterile RPMI) or peritoneal lavage fluid (S1 Fig). Using RT-PCR we determined the levels of histidine decarboxylase (HDC) from whole blood RNA. In contrast to histamine, blood levels of HDC mRNA increased throughout the 12 weeks, indicating that histamine may be continually synthesized during infection (Fig 1B). Using ELISA, we determined circulating levels of Ls-specific IgE. Ls-specific IgE levels became detectable after 4 weeks of infection (Fig 1C). Development of detectable Ls-specific IgE thus preceded peak histamine levels in the plasma, suggesting peak histamine release may be due to IgE-mediated basophil and mast cell activation. In contrast, the immediate early release of histamine at the 30 minute time point is suggestive of parasite-specific antibody independent activation of basophils and mast cells. We next sought to determine whether histamine plays a role in maintaining worm burdens during primary infection. To test this, BALB/c mice were infected with 40 L3s and treated with HR1 or HR2 antagonists administered in water for the duration of infection. At 8 weeks, mice were euthanized and adult worm burden was determined. Untreated infected mice had a mean recovery of 18 adult worms. Mice treated with HR1 antagonists had a mean recovery of 8 adult worms (58. 1% reduction, p = 0. 001) while mice treated with HR2 antagonists had a mean recovery of 13 worms (22. 5% reduction, p = 0. 0573) (Fig 2). This data indicates that signaling via HR1 may play a role in long-term survival of L. sigmondontis in the mammalian host. Given that we observed a reduction of adult worms at 8 weeks post-infection in animals treated with an HR1 antagonist, we sought to determine if HR1 antagonism altered the circulating microfilaria load or the male-to-female ratio. HR1i administration did not alter the number of microfilariae circulating in the blood (S2 Fig) or the male-to-female ratio of recovered adult worms (S3 Fig). The lack of a decrease in microfilaria burden is not too surprising as microfilaria load does not correlate with adult worm numbers [13]. Due to the observed reduction in adult worm burdens at 8 weeks, we next sought to determine whether there was a particular timepoint during infection when HR1 blockade enhances worm clearance. In a typical course of infection, L3 stage larvae migrate from the subcutaneous tissues to the pleural space from days 1–5, molt to L4 stage worms by d 11, and then molt to adult worms between days 24–30. Mice were treated for 10,35, and 56 days post-infection with HR1 antagonist. At 56 days (8 weeks), all groups of mice were euthanized and living adult worms collected. Recovered worms that were motile yet had parts that were covered with granulomas were classified as “encased” (Fig 3A and 3B). As previously demonstrated, mice treated with HR1 antagonists for 8 weeks demonstrated a significant reduction in adult worm burden compared to untreated mice (Fig 3C). While there was no difference in total worm burden at the 8 week timepoint between mice treated with HR1 antagonists for 10 days and untreated mice (mean worm burden of 17 vs 20), mice treated with 10 days of fexofenadine (HR1i) had significantly greater numbers of adult worms that were encased in granulomas (Fig 3D) at 56 days post infection. The trend towards lower worm burdens with longer fexofenadine treatment courses suggests that H1R blockade enhances worm clearance at numerous stages of worm development. Given the reduction in adult worm burden observed in HR1 treated mice, we next evaluated whether fexofenadine is directly toxic to L. sigmodontis worms and whether exogenous histamine enhances worm survival. To test this, L3 stage worms were cultured in vitro and supplemented daily with 200nM histamine, 10mM of fexofenadine, or media alone and assessed daily for survival. As seen in Fig 4, there were no observed differences in survival times between L3s supplemented with histamine, L3s supplemented with fexofenadine, and those supplemented with media (Fig 4). These data indicate that exogenous histamine does not directly enhance worm survival and that fexofenadine is not directly toxic to worm viability. Because fexofenadine did not appear directly toxic to L. sigmodontis worms, we next evaluated whether H1R antagonism alters the immune response that develops during infection. To assess this, humoral and cellular immunological studies were conducted on infected mice treated with 8 weeks of fexofenadine. Both total and LsAg-specific IgE were significantly decreased in mice treated with fexofenadine compared to untreated controls (Fig 5A and 5B). In terms of cellular immune responses, parasite antigen-driven production of IL-5 and IFN-γ from splenocytes was also significantly reduced in fexofenadine treated mice (Fig 5D and 5E), whereas IL-4 production was not (Fig 5C). This suggests that signaling via HR1 may enhance both type 1 and type 2 immune responses. In contrast to the decreases in IgE, IL-5, and IFN-γ, the cellular infiltrate present in the pleural cavity, the site where adult L. sigmodontis worms reside, was dramatically increased in fexofenadine treated mice. Whereas infection of untreated BALB/c mice resulted in a median of 1. 7 x 106 cells in the pleural space at study endpoint, fexofenadine-treated mice had 4. 8 x 106 cells (p = 0. 0080, Fig 6A). Flow cytometric analysis revealed that eosinophils comprised over half of the cells in the pleural infiltrate of fexofenadine-treated mice, increasing in numbers from a median of 4. 7 x 105 cells in untreated infected mice to 2. 5 x 106 cells fexofenadine-treated infected animals (p = 0. 0043, Fig 6B) A number of studies utilizing the L. sigmondontis model have demonstrated a significant role for eosinophils in immune-mediated clearance of worms [14,15]. As fexofenadine increased eosinophil numbers at the site of adult worm infection, we next tested whether fexofenadine mediated worm clearance is dependent on eosinophils. Eosinophil deficient mice (ΔdblGATA) and background control BALB/c mice were infected with L. sigmondontis, treated for 8 weeks with HR1 antagonists, and euthanized at 8 weeks for enumeration of adult worm burden. In contrast to fexofenadine-treated wild type mice, eosinophil deficient mice administered fexofenadine had no reduction in adult worm burden (mean recovery 18) when compared to untreated ΔdblGATA controls (mean recovery 15) or BALB/c background controls (mean recovery 14) (Fig 7). To further assess the activity of eosinophils in antihistamine mediated worm clearance, BALB/c mice were infected, treated with HR1 antagonists for 8 weeks, and then euthanized. A pleural lavage was performed and ELISA used to detect eosinophil peroxidase (EPO) as evidence of eosinophil degranulation. Fexofenadine treated mice demonstrated significantly highler levels of EPO in the lavage fluid (S4 Fig). Taken together, these findings demonstrate that H1R blockade enhances worm clearance through an eosinophil-dependent mechanism. In this study we found that histamine is released throughout filarial infection, that antihistamine therapy reduced IgE levels and increased eosinophilic responses at the site of infection, and that administration of fexofenadine, a HR1 blocker, enhances clearance of adult worms in an eosinophil-dependent manner. Our first experiment was a time course study of circulating histamine levels to determine the kinetics of histamine release during primary filarial infection. As the t1/2 of histamine in blood is approximately 60s [16,17], blood levels are representative of ongoing histamine release. We found that histamine was released throughout the course of primary L. sigmondontis infection. The 1st peak in circulating histamine occurred 30 minutes post infection in naïve mice. This finding has two important implications. First, as basophils and mast cells are the only cells carrying pre-formed histamine [18], it suggests that one or both of these cell types are activated within minutes of filaria infection. Early activation of these cells may be important for the shaping of the immune response to tissue invasive helminths. Second, early histamine release represents a non-specific mechanism of mast cell or basophil activation, since specific IgE is not present until weeks after infection. This is consistent with a number of studies that have demonstrated direct activation of basophils by helminth antigens (reviewed in [10]). Timecourse studies next revealed that histamine release in infected mice peaks again at 8 weeks of infection. We speculate that the 8 week peak may be due to basophil and mast cell activation in response to circulating microfilariae, which appear starting 7 weeks post-infection. As detectable LsAg-specific IgE develops by 6 wks post-infection, this activation is likely occurring through IgE. This 2nd peak is then followed by a decrease in circulating histamine, even though histamine decarboxylase message in blood cells increases throughout infection. This data is consistent with previous findings that basophils become hyporesponsive over time, requiring more signal to achieve activation [19]. Therefore, even though histamine synthesis continues throughout the course of infection, basophils and mast-cells are releasing less histamine in the chronic stages of infection. Perhaps the most striking finding of this study is the significant reduction of adult worm burden at 8 weeks in mice treated with a HR1 antagonist. To determine the timing of worm clearance, infected mice were treated for 10,35, or 56 days with fexofenadine and assessed for adult worm burden at 8 weeks. We found that the longer mice were treated with fexofenadine the greater the reduction in adult worm burden at 8 weeks and that treatment with fexofenadine resulted in a significant increase in encased worms in all groups. Previous studies have suggested a role for histamine in early larval invasion into the lymphatics [20]. Taken together these data indicate that that HR1 blockade enhances worm clearance at numerous stages of development. As in vitro studies revealed that fexofenadine is not directly toxic to Litomosoides sigmodontis, we next evaluated whether fexofenadine augments immune responses directed against the parasite. Although helminth-specific IgE, IL-5 and IFNγ responses were all decreased in fexofenadine treated mice, eosinophil numbers at the site of worm infection were significantly elevated. Experiments with eosinophil deficient ΔdblGATA mice demonstrated that eosinophils were required for fexofenadine-mediated helminth clearance. In contrast to fexofenadine treated wild type mice, which had 80% fewer adult worms than wild type controls, fexofenadine treated ΔdblGATA mice exhibited no decrease in worm numbers. These results are consistent with prior studies suggesting eosinophils are key effector cells against helminths [21–23]. Studies utilizing the L. sigmondontis model have shown that mice deficient in eosinophil peroxidase or major basic protein, key eosinophil granule proteins, have significantly higher filarial worm burdens than wild type controls [14]. Prior studies [24] have shown that mice deficient in IL-5 produce neutrophilic rather than predominantly eosinophilic granulomas around L. sigmodontis worms. In our study, IL-5 production was likely not the driving mechanism for larger eosinophilic granulomas in the setting of H1R blockade as splenocytes from fexofenadine-treated mice demonstrated less IL-5 production than splenocytes from untreated infected animals. Together, the results of these papers and this study suggest that IL-5 is required for eosinophilic granuloma formation, and that fexofenadine enhances this process. There are multiple mechanisms by which HR1 blockade may have enhanced eosinophil responses in this study. One possibility is that HR1 blockade may have enhanced eosinophil survival. Data from one in vitro study suggests histamine signaling reverses IL-5 afforded eosinophil survival [25]. A second hypothesis to explain increased eosinophil numbers at the site of worm infection is enhancement of eosinophil chemotaxis by blockade of HR1 signaling. Histamine is a known chemoattractant molecule for eosinophils [26–28], and the recently discovered [29] histamine receptor 4 (HR4) has been demonstrated to play a significant role in eosinophil chemotaxis and activation [30–35]. As such, it is possible that blockade of histamine signaling through HR1 enhances the effects of histamine through HR4. Alternatively, HR1 blockade may indirectly enhance eosinophil chemotaxis by increasing production of eosinophil chemotaxins or augmenting eosinophil sensitivity to such agents. Of note, dblGATA mice are deficient in basophils as well as eosinophils. Thus, it is possible that worm clearance in fexofenadine-treated mice is due to the action of basophils rather than eosinophils. However, as we have previously found that depletion of basophils does not alter adult worm numbers ([36], [37]), and as eosinophils are known to have the ability to kill adult filarial worms [14,38], we believe it is most likely that worm clearance induced by fexofenadine is through enhancement of eosinophil numbers at the site of infection. One of the most interesting findings of this study is the observation that fexofenadine treatment caused significant reductions in circulating IgE levels and splenocyte production of IL-5 and IFNγ as well as increased numbers of eosinophils at the site of infection. While we can only speculate on the mechanisms underlying these apparently contrasting findings, we expect that it may be related 1) to the concentrations of histamine locally (at the site of infection) vs systemically, and 2) to unknown effects of histamine on the function of various immune effector cells. Another possibility is that decreased IgE levels and IL-5 production may have been due to the decreased adult worm burdens observed in fexofenadine-treated mice. The concentrations of histamine at different body sites during infection and the effects histamine has type 2 responses from B cells, T cells, macrophages, and dendritic cells will be the focus of future investigations. Another possibility is that decreased IgE levels and IL-5 production may have been due to the decreased adult worm burdens observed in fexofenadine-treated mice. These findings demonstrate that histamine, in addition to its immediate proinflammatory effects, also functions to shape the immune response to helminth infections. The exact mechanisms by which this occurs are not yet clear. Histamine is known to alter the immunological function of a variety of cell types, including epithelial cells, granulocytes, T-cells, B-cells, and dendritic cells [3,39–41]. Investigations combining HR1 deficient mice with airway hyperresponsiveness models are mixed [42–45]. Whereas one showed decreases in type 2 cytokines, no changes in IFNγ, decreased IgE levels, and increased blood eosinophil numbers [42], another showed increases in type 2 cytokines, decreased IFNγ, and decreased bronchoalveolar lavage eosinophil numbers [43]. We believe the differences in these studies demonstrate the complex role histamine plays in shaping immune responses. The exact effects of histamine are likely dependent not only on the cell types involved, but also on the cytokine environment in which histamine is acting and on the repertoire of histamine receptors displayed by individual cells. The results of our study may have some important clinical ramifications. Currently there is a worldwide effort to control and potentially eradicate lymphatic filariasis and onchocerciasis by repeated mass drug administration (MDA) of anti-filarial medications, especially diethylcarbamazine (DEC) [44]. A major factor limiting success of MDA is the inability of anti-filarial drugs to kill adult worms when given as a short course [45]. Since antifilarial medications primarily clear microfilariae, ongoing mass drug administration programs require repeated administration of antifilarial agents s for years until natural death of adult worms occurs. [46,47]. One of the interesting aspects of DEC therapy is that DEC does not appear sufficient on its own to kill filarial worms. Numerous studies have shown that DEC-mediated clearance of filariae is dependent in large part on the host immune response [48,49]. Since we have shown that fexofenadine can augment immune clearance of adult filarial worms, we hypothesize that addition of fexofenadine to DEC or other antifilarial medications may result in better adult worm eradication than current regimens. Discovering a short course therapy that can successfully eliminate adult filarial worms would greatly increase our ability to control and eradicate filarial infections. Further elucidating the mechanisms by which fexofenadine decreases adult worm burdens, and investigating whether combining fexofenadine with current antifilarial medications enhances adult worm clearance, will be the focus of future studies. | Title: Histamine 1 Receptor Blockade Enhances Eosinophil-Mediated Clearance of Adult Filarial Worms Summary: Filariae are tissue-invasive parasitic roundworms that infect over 100 million people worldwide and cause debilitating conditions such as river blindness and elephantiasis. One of the major factors limiting our ability to eliminate these infections is the lack of drugs that kill adult worms when given as a short course therapy. Additionally, the mechanisms by which adult worms are cleared from infected individuals remains poorly understood. In this study, we demonstrate that treatment of infected mice with fexofenadine, an inhibitor of histamine receptor 1, significantly reduces adult worm numbers through a mechanism dependent on host eosinophils. These findings suggest that histamine release induced by parasitic worms may aid parasite survival by decreasing eosinophilic responses. Further, as antihistamines are generally safe medications, these results raise the possibility that antihistamine therapy may be useful either alone, or potentially in combination with other antifilarial medications such as diethylcarbamazine (DEC), to eliminate adult filarial worms from infected individuals. | 6,470 | 246 | lay_plos | en |
Write a title and summarize: Combining clonal analysis with a computational agent based model, we investigate how tissue-specific stem cells for neural retina (NR) and retinal pigmented epithelium (RPE) of the teleost medaka (Oryzias latipes) coordinate their growth rates. NR cell division timing is less variable, consistent with an upstream role as growth inducer. RPE cells divide with greater variability, consistent with a downstream role responding to inductive signals. Strikingly, the arrangement of the retinal ciliary marginal zone niche results in a spatially biased random lineage loss, where stem- and progenitor cell domains emerge spontaneously. Further, our data indicate that NR cells orient division axes to regulate organ shape and retinal topology. We highlight an unappreciated mechanism for growth coordination, where one tissue integrates cues to synchronize growth of nearby tissues. This strategy may enable evolution to modulate cell proliferation parameters in one tissue to adapt whole-organ morphogenesis in a complex vertebrate organ. To maintain proper proportions, growth must be regulated at the level of the whole body, the size of each organ, and the size of tissues within an organ (Roselló-Díez and Joyner, 2015). Some regulatory mechanisms are shared, while others are specific to each level or to particular organs (Lui and Baron, 2011; Roselló-Díez and Joyner, 2015). Systemic signals couple nutrition to growth to coordinate growth of all organs at the organismal level (Buchmann et al., 2014; Droujinine and Perrimon, 2016). In addition to extrinsic systemic factors, transplantation experiments showed that many organs, including the eye, grow autonomously according to intrinsic factors (Wallman and Winawer, 2004; Roselló-Díez and Joyner, 2015). Growth coordination mechanisms have been studied at the level of the whole organism and inter-organ communication (Buchmann et al., 2014; Droujinine and Perrimon, 2016), but feedback mechanisms between constituent tissues of an organ remain largely unexplored both experimentally and at a conceptual level (Buchmann et al., 2014). Teleost fish grow throughout their lives, increasing massively in size (Johns and Easter, 1977). The teleost medaka (Oryzias latipes) grows roughly ten-fold from hatching to sexual maturity within 2–3 months (Figure 1—figure supplement 1A). Unlike embryonic morphogenesis, during post-embryonic growth all organs must scale with the increasing body size while fully functioning. In the eye, continuous growth must be additionally balanced with continuous shape-keeping: Proper optics, and thus vision, requires a precise 3D shape. Highly visual shallow water fish such as medaka have near-perfect hemispherical eyes (Fernald, 1990; Nishiwaki et al., 1997; Beck et al., 2004). The growth rates of all eye tissues must perfectly match, otherwise the organ would deform, akin to a bimetallic strip. Thus, the eye of fish provides an excellent system to explore how anatomically and functionally distinct tissues coordinate to grow and maintain the shape of an organ in functional homeostasis (Johns and Easter, 1977; Centanin et al., 2014). The vertebrate eye consists of multiple concentric tissues, including the neural retina (NR) and the retinal pigmented epithelium (RPE) (Figure 1A; Table 1). In fish and amphibians, these tissues grow from a ring-shaped stem cell niche in the retinal periphery: the ciliary marginal zone (CMZ) (Johns, 1977; Harris and Perron, 1998; Amato et al., 2004). The CMZ can be subdivided into a peripheral stem- and a central progenitor cell domain; stem cells are believed to have the potential for indefinitely many cell divisions while progenitor cells divide only a handful of times (Raymond et al., 2006; Centanin et al., 2014; Wan et al., 2016; Shi et al., 2017). At the very periphery of the CMZ, about 5 rows of cells express the stem cell marker retina-specific homeobox gene 2 (Rx2) (Reinhardt et al., 2015; Wan et al., 2016; Tang et al., 2017). The CMZ is a bi-partite niche, with tissue-specific stem cells for NR and RPE (Shi et al., 2017). In medaka, stem cells for NR and RPE are strictly separate, as demonstrated by transplantations at blastula stage and genetic recombination after hatching (Centanin et al., 2011; Centanin et al., 2014). Thus, medaka NR and RPE are independently growing tissues with identical topology. As a population, CMZ cells appositionally add new cells in concentric rings as shown by label incorporation with thymidine analogues (Johns, 1977; Centanin et al., 2011). Individual stem cells labelled by genetic markers form clonal progeny in so-called Arched Continuous Stripes (ArCoS; Figure 1B) (Centanin et al., 2011; Centanin et al., 2014). Medaka NR stem cells produce the full complement of neuronal cells in apico-basal clonal columns (Figure 1—figure supplement 2A’–B) (Centanin et al., 2011; Centanin et al., 2014; Lust and Wittbrodt, 2018). These differentiated retinal cells grow little in size (Johns, 1977), retain their relative position over time (Johns, 1977; Centanin et al., 2011), and have negligible death rates (Johns and Easter, 1977; Stenkamp, 2007). Thus, the only parameter available to NR and RPE to coordinate their growth rates is the proliferation of the tissue-specific CMZ stem cells. Stem cells have long been defined by an unlimited self-renewal capacity (Watt and Hogan, 2000; Clevers and Watt, 2018). Two general strategies underlie long-term maintenance of stem cells: 1) a deterministic model where every single division produces a stem- and a progenitor daughter cell (‘invariant asymmetry’); and 2) a stochastic model where cells divide symmetrically, and the daughter cells have a probability to stay as stem cells or commit to a progenitor fate (‘neutral drift’) (Watt and Hogan, 2000; Clevers and Watt, 2018). One tenet of this model is neutral competition: Stem cells randomly displace one another, resulting in the ‘loss’ of lineages where all progeny commit to a progenitor fate until the entire niche is occupied by a single clone (Colom and Jones, 2016; Clevers and Watt, 2018). Strikingly, the medaka retina diverges from the neutral drift model. The CMZ maintains a polyclonal stem cell population for both the NR and the RPE, and in particular NR stem cells undergo asymmetric self-renewing divisions throughout the life of the animal (Centanin et al., 2011; Centanin et al., 2014). It remains unclear whether stem cell proliferation in the CMZ follows a purely deterministic model, or whether it follows a strategy in-between invariant asymmetry and neutral drift. In this work we combine in vivo and in silico clonal analysis in the NR and RPE of medaka to address how these tissues coordinate their growth rates. We find that RPE stem cells have highly variable cell division timing consistent with a downstream role in the control hierarchy, whereas NR stem cells display less variability consistent with an upstream role in inducing growth in nearby tissues. Our simulation predicts that the spatial segregation of stem and progenitor CMZ domains is an emergent property, as the topology of the retinal niche preconditions the retina to a spatially biased neutral drift. NR stem cells deviate from a purely random drift model by preferential division axis orientation and differential modulation of division parameters along the CMZ circumference. We propose that during post-embryonic growth of the teleost eye, the NR CMZ forms a hub for integrating external and internal stimuli that affect cell division parameters, which ultimately direct the growth and shape of the entire eye. Retinal cells follow an exquisite spatiotemporal order (Figure 1B–C, Figure 1—figure supplement 1B). Thus, clones derived from stem cells are a frozen record of past cell divisions (Centanin et al., 2011; Centanin et al., 2014), offering a window of opportunity to study stem cell properties in the NR and RPE. We experimentally generated NR ArCoS by randomly labelling individual NR stem cells using the Rx2: : ERT2Cre, Gaudí2. 1 line in hatchling medaka, and analyzing the eyes in adult fish as previously described (Centanin et al., 2014; Reinhardt et al., 2015). The Rx2 promoter drives the inducible Cre recombinase in stem cells at the very periphery of the CMZ (Reinhardt et al., 2015). A recombined stem cell generates a stripe of GFP-positive progeny in an otherwise GFP-negative retina (Centanin et al., 2014). In proximal view, NR ArCoS emanated as rays from the central embryonic retina, the part of the eye that was already differentiated at the timepoint of Cre-mediated recombination (Figure 1D). We visualized RPE ArCoS by mosaic knockout of pigmentation using CRISPR/Cas9 targeted to the gene oculo-cutaneous albinism 2 (Oca2), which is required for melanosome maturation (Fukamachi et al., 2004; Lischik et al., 2019). RPE stem cells with a bi-allelic mutation in Oca2 generate unpigmented stripes, analogous to RPE ArCoS obtained by transplantation (Centanin et al., 2011). RPE ArCoS frequently branched, forming irregular stripes variable in size and shape (Figure 1E). These qualitative differences in clonal pattern suggested that despite their identical topology, the division behavior of NR and RPE stem cells differed. Clonal data generates a distribution of outcomes that is challenging to analyse and easy to misinterpret (Klein et al., 2007). The curved retinal surface and spatial extent of the niche pose a further challenge. We overcome these challenges by comparing experimental clonal data with simulated clonal data from a 3D agent based cell-center overlapping spheres model built in the platform EPISIM (Sütterlin et al., 2013; Sütterlin et al., 2017; Sütterlin, 2019). This modelling technique represents cells as discrete objects (e. g. spheres) that physically interact through forces acting on the cell centers; the spheres are allowed to slightly overlap to simulate cell deformability and allow a tight cell packing (Sütterlin et al., 2013; Sütterlin et al., 2017). This level of abstraction is ideally suited to the tightly packed pseudocrystalline mosaic of retinal cells (Johns, 1981; Nishiwaki et al., 1997; Pérez Saturnino et al., 2018), and has been used previously to model clonal data in skin and gut epithelia (Osborne et al., 2010; Buske et al., 2011; Li et al., 2013). Our retinal tissue model consists of a layer of spheres (representing either NR or RPE cells) on a hemisphere (representing the rest of the organ that is not explicitly modelled; Figure 2A). The RPE is a monolayer, thus each model cell corresponds to one RPE cell. In the NR, CMZ stem cells form a monolayer, and their differentiated progeny arrange in multiple neuronal layers (Johns, 1977; Raymond et al., 2006). We observed that clonal progeny of CMZ stem cells retained close proximity with little spread tangential to the retinal surface, forming clonally related ‘columns’ (Figure 1—figure supplement 2A’-B) (Centanin et al., 2011; Centanin et al., 2014; Lust and Wittbrodt, 2018). We took advantage of this fact to abstract each differentiated clonal column as a single cell in the simulation. In vivo, the spatial extent of the CMZ stem cell domain is believed to be defined by cues such as nearby blood vessels (Wan et al., 2016; Tang et al., 2017). Therefore, we defined the virtual stem cell domain with a fixed size of 25 µm, that is 5 rows of cells, reflecting the endogenous scale of the Rx2-expressing CMZ domain (Reinhardt et al., 2015; Wan et al., 2016; Tang et al., 2017). In vivo, NR stem cells divide predominantly asymmetrically, but also undergo symmetric divisions (Centanin et al., 2014). The rates of asymmetric and symmetric divisions are unknown; likewise, it is unknown whether these rates are deterministically defined or an emergent property of an underlying stochastic system. Since stochastic cell divisions successfully describe the proliferation of committed retinal progenitor cells in larval zebrafish (Wan et al., 2016), we used a simple stochastic mechanism for our initial model. Virtual stem cells commit to divide with a fixed probability pdivision=126h−1 and intervals between subsequent cell divisions must fulfill a minimum cell cycle length tcellCycle=24h. These values lie within a biologically plausible range estimated from experimentally measured growth rates and a parameter scan of the simulation (Appendix 1 section 3. 3). All divisions are symmetric, resulting in two stem cells; cells differentiate and stop cycling when they exit the virtual CMZ after being pushed out by cellular crowding. To prevent physically implausible cell crowding, cell-center based models include a density-dependent inhibition of cell division (Pathmanathan et al., 2009; Osborne et al., 2017; Sütterlin et al., 2017). In our model, inhibition occurs in cells whose average overlap with all neighbors exceeds a fraction of the cell’s diameter given by the model parameter δol_threshold (Figure 2—figure supplement 1; Appendix 1, section 2. 4). Based on in vivo observations (Lyall, 1957; Johns, 1977; Ohki and Aoki, 1985), the growing virtual eye gradually moves cells apart as it expands, thus decreasing cell density (Figure 2—figure supplement 2; Appendix 1 section 2. 2). Continuous proliferation in the CMZ counteracts this decrease in vivo (Johns, 1977; Johns and Easter, 1977); likewise, the ever-increasing virtual cell population optimally fills the hemisphere at all times (Video 1; Video 2). Our model distills the complexity of the system and replicates the exquisite spatiotemporal growth order observed in vivo (Figure 2B’, B’’). Conceptually, we reasoned that feedback between tissues in an organ can be wired in two fundamental ways: Either the tissue of interest acts upstream to induce growth of other tissues (Figure 2C’; ‘inducer growth mode’), or, vice versa, the tissue of interest lies downstream of growth cues from another tissue in the organ (Figure 2D’; ‘responder growth mode’). Possible biological mechanisms for these growth modes could be mechanical, biochemical, or a combination of both. For example, in the inducer growth mode cells could instruct organ growth by modifying the extracellular matrix or by paracrine signalling (Buchmann et al., 2014; Droujinine and Perrimon, 2016). These stimuli instruct tissues with the responder growth mode to grow, for example by alleviating contact inhibition or by providing permissive proliferation signals (Buchmann et al., 2014; Droujinine and Perrimon, 2016). In an organ composed of multiple tissues, one tissue may be the driver for growth, while the rest follows. We examined how these two conceptual growth modes affected stem cell dynamics in the simulation. In our implementation of the inducer growth mode, an increase in cell number induces growth of the virtual eye’s radius (Appendix 1—equation (5) ). Implicit in this growth mode is the assumption that cell division is not inhibited by the degree of cell crowding normally present in the tissue (otherwise the organ would never grow). Therefore, we set the tolerated overlap threshold δol\_threshold=0. 4, a value which we determined by parameter scan to minimize cell division inhibition while preventing physically implausible crowding (Appendix 1, section 3. 2). In the responder growth mode, we let the radius grow linearly over time (Appendix 1—equation (6) ). In this growth mode, cells must stop dividing until they receive an external stimulus. We take advantage of the pre-existing local density sensing to implement a physical stimulus akin to contact inhibition. Thus, we set the tolerated overlap threshold δol\_threshold=0. 2 to maximize cell division inhibition at homeostatic density (Appendix 1, section 3. 2). As growth of the hemisphere decreases cell density, cells dynamically respond to growth of the eye by resuming divisions. In short, the growth modes in our simulation differ only in: 1) the growth equation for the radius of the hemisphere, 2) the value of the threshold parameter δol\_threshold where local cell density inhibits cell divisions (for details, the reader is referred to Appendix 1, sections 2. 3; 2. 4; and 3. 2). We obtained virtual ArCoS regardless of growth mode (Figure 2C’’, D’’). The growth mode strongly impacted on the shape of ArCoS. Clones in the inducer growth mode formed well-confined stripes with low variation in shape (Figure 2C’’’). In the responder growth mode, the virtual clones frequently intermingled and broke up into smaller clusters (Figure 2D’’’). Specifically, the growth modes impacted on variation in cell division timing (Figure 2C’’’’, D’’’’). In the responder growth mode, local competition for space increased cell division intervals, particularly among cells exceeding the tolerated overlap threshold δol\_threshold=0. 2 (Figure 2D’’’’). Thus, the model predicted distinct levels of variation in cell division timing in retinal tissues following the inducer or responder growth modes. Since the position of cells in the retina reflects their birth order (Centanin et al., 2011; Centanin et al., 2014), we reasoned that in the extreme case of no variation in cell division timing, each clone forms a continuous, unbranching stripe (Figure 3B, left). In the opposite highly variable case, clones frequently branch or merge into polyclones, as well as fragment into several small patches (Figure 3B, right). Thus, with increasing variation in cell division timing, we expect an increasing variation in clone width, and an increasing incidence of clone branching and fragmentation. To quantitatively underpin our previous observations, we compared simulated clones of the inducer and responder growth modes to clones in the NR and RPE (Figure 3A’, A’’). We circumvented biases associated with fusion and fragmentation of clones by analyzing ‘patches’, that is contiguous domains of segmented pixels. A patch may entail a (sub-) clone, or multiple clones (i. e. a polyclone) (Figure 3—figure supplement 1; Video 3). To assay our experimental and simulated data, we unrolled the retina with a coordinate transform (Figure 3—figure supplement 2C) and quantified three different metrics: patch width variance, branching, and fragmentation. To assay patch width variance, we aligned and superimposed all patches (Figure 3C’, C’’), and quantified the distribution of maximum patch width (Figure 3—figure supplement 2A; Figure 3—figure supplement 2—source data 3.). Confirming our previous qualitative observations, NR patches formed a narrow stripe, while the width of RPE patches showed much greater variation (Figure 3C’; Figure 3—figure supplement 2A). The variance of NR and RPE patches was significantly different at the 0. 05 level (p=3. 50∙10−12, F-test of equality of variance). In striking agreement to the experimental data, simulated patches in the inducer growth mode had low variation in width, while patches in the responder growth mode spread widely (Figure 3C’’; Figure 3—figure supplement 2A). The variances in the simulated conditions were significantly different at the 0. 05 level (p=5. 84∙10−7, F-test of equality of variance), but highly similar between NR and inducer (p=0. 56, F-test of equality of variance); and RPE and responder (p=0. 21, F-test of equality of variance). To measure branching we skeletonized the patches, and quantified the distribution of nodes per patch and condition (Figure 3D; Figure 3—source data 5). Patches in the NR and in the inducer growth mode were overwhelmingly stripe-like with no branch points (Figure 3D; inset I), with similar node distribution (p=0. 64, Wilcoxon rank sum test). In contrast, both NR and inducer differed significantly at the 0. 05 level from the distribution in the RPE and responder growth mode (NR-RPE: p=3. 93∙10−6; NR-responder: p=3. 26∙10−4; inducer-RPE: p=6. 24∙10−7; inducer-responder: p=7. 00∙10−5, Wilcoxon rank sum test). Patches in the RPE and in the responder growth mode frequently bifurcated or merged, creating branching shapes with inclusions indicative of clone intermingling (Figure 3D; inset III). RPE and responder growth mode were highly similar in this metric (p=0. 38, Wilcoxon rank sum test). Not all patches were contiguous with the embryonic retina. Such ‘late arising patches’ result if a cell divided intermittently with periods of dormancy, leaving clone fragments behind (Figure 3B, highly variable scenario). We quantified fragmentation by plotting the occurrence of late arising patches along the normalized post-embryonic retinal radius (Figure 3E; Figure 3—source data 6). In the NR late patches clustered in the central post-embryonic retina and waned thereafter. Thus clone fragments were not equally distributed, consistent with lower levels of cell division variability and a majority of continuous stripe-like clones. In contrast, the RPE displayed an even distribution indicative of frequent fragmentation throughout the life of the animal as predicted for the highly variable scenario (NR-RPE: p=1. 74∙10−3, Wilcoxon rank sum test). The simulated data showed the same tendency, to a lesser degree, as the central peak in late patches was higher in the inducer growth mode and peripheral late patches occurred more frequently in the responder growth mode (Figure 3E; inducer-responder: p=0. 10, Wilcoxon rank sum test). In this metric, the RPE stood out from the NR and both simulated conditions (RPE-inducer: p=6. 94∙10−5; RPE-responder: p=0. 04, Wilcoxon rank sum test), indicating a high degree of fragmentation and thus cell division variability. Together, these data show that NR and RPE have different degrees of variability in cell division timing. The NR displayed lower variability consistent with the simulated inducer growth mode, while the RPE showed higher levels of variability that even exceeded what we modelled with the responder growth mode. Thus, our data support a model where NR and RPE concertedly expand relying on different growth modes, which manifest in differently shaped ArCoS. Both the NR and simulations displayed a cluster of late patches in the central post-embryonic retina (Figure 3E). Additionally, when discounting late patches, the distribution of patch length showed clear bimodality (Figure 3—figure supplement 2B), suggesting that beyond fragmentation an additional stochastic process took place after clonal labelling. The region at the border to the embryonic retina, the ‘induction ring’, marks the original position of the CMZ at the timepoint of Cre-mediated recombination (Figure 4E). To investigate the stem cell dynamics in the induction ring we turned to the simulation. Surprisingly, the virtual induction ring contained many few-cell clones unrelated to any ArCoS (Figure 4A’, encircled by pink dashed lines). In these clones, all stem cells left the niche and thus differentiated (‘terminated clones’). Nested inductions showed that sister stem cells within one clone segregated into subclones (Figure 4A’–A’’, highlighted ArCoS). However, only some of these subclones generated virtual ArCoS. Again, terminated clones clustered in the virtual induction ring (Figure 4A’’, encircled by black dashed lines), demonstrating that the pattern repeated itself regardless of the timepoint of virtual induction. Therefore, since central positions were occupied by short terminated clones, many stripe-like patches necessarily began in more peripheral positions, explaining the peak in late arising patches. In our model, all proliferative cells were equipotent stem cells. Nevertheless, a subset of these virtual stem cells proliferated only a few times before terminally differentiating, resulting in a bimodal distribution of patch lengths (Figure 3—figure supplement 2B). Notably, the overwhelming majority of virtual ArCoS emerged from the periphery of the induction ring (Figure 4A’–A’’; Video 4), as confirmed by tracing back the position of the founder stem cells at simulation step 0, while centrally located cells formed exclusively terminated clones (Figure 4B). This behavior is highly reminiscent of retinal progenitor cells in vivo, which are believed to reside in the central CMZ (Raymond et al., 2006; Shi et al., 2017). Strikingly, only a minority of virtual stem cells formed ArCoS, while the vast majority formed terminated clones (Figure 4B). Together, these data show that the virtual stem cell population subdivided into two functional domains that mirror the current model of the retinal niche with a peripheral stem- and a central progenitor domain (Raymond et al., 2006; Shi et al., 2017). Importantly, this subdivision was not imposed onto the simulation, but emerged dynamically. The central-most cells were poised to differentiate by being pushed out of the niche by divisions of their more peripheral neighbors. This neutral competition occurred continuously, as demonstrated by nested virtual inductions (Figure 4A’–A’’). Thus, the spatial segregation of stem- and progenitor domains is an emergent property of the system. Our simulations uncovered a role of stochastic drift in the niche, and lead us to the following two predictions: First, a large proportion of stem cells is lost by neutral competition and forms terminated clones. Thus, ArCoS should be a minority among labelled clones. Second, there is a spatial bias in this drift: The majority of ArCoS will derive from peripheral cells but some will derive from more central positions. Similarly, the majority of terminated clones will derive from central positions, but some will derive from peripheral positions. To address these predictions experimentally, we again labelled NR stem cells in hatchlings using the Rx2: : ERT2Cre, GaudíRSG line (Centanin et al., 2014; Reinhardt et al., 2015), which when recombined results in a nuclear GFP signal, and analysed the eyes at adult stage. Few-cell clusters in the induction ring vastly outnumbered ArCoS, showing that terminated clones were the most common type of clone (n = 1129 terminated clones in 20 retinae; Figure 4C’–C’’, Figure 4—figure supplement 1A–B). A small fraction of terminated clones extended into the post-embryonic retina (Figure 4C’–C’’, yellow arrowheads). ArCoS, which by definition always reach the retinal margin, were less frequent (Figure 4C’–C’’, pink arrowheads; n = 36 ArCoS in 20 retinae). Thus, Rx2-expressing cells in the CMZ included cells that proliferated indefinitely as well as cells that proliferated only a few times before differentiating. The preponderance of terminated clones shows that ArCoS-forming cells are a minority, in line with our first prediction. To address the spatially biased stochastic drift, we examined at which position in the induction ring clones contained their central-most pixels in experiment and simulation (Figure 4C’–C’’, D’–D’’, F). Among terminated clones, the majority started in central positions (experiment: 77. 3%; simulation: 61. 0%), while a minority were exclusively located in the peripheral induction ring or in the post-embryonic retina (experiment: 22. 7%; simulation: 39. 0%). The difference in proportions between experiment and simulation may indicate that the simulation underestimates the number of terminated clones. Nevertheless, a sizeable subset of experimental terminated clones derived from the periphery of the stem cell domain of the CMZ, indicating that some stem cells drifted into a progenitor-like state. Among experimental ArCoS, the vast majority (86. 1%) started in the periphery, but 13. 9% derived from a central position, showing that some cells located in the central progenitor domain of the CMZ drifted into a lifelong stem cell fate. Strikingly, the ratios for peripheral and central ArCoS in the simulation are nearly identical (p=1. 00,2-sample test for equality of proportions), showing that the simulation captures ArCoS dynamics extremely well. Together, these data support a model of stochastic drift with a peripheral-stem and central-progenitor bias that is conditioned by the physical topology of the niche. NR ArCoS formed stripes that appeared slightly narrower than in the simulation (Figure 3A’–A’’, C’–C’’). In simulations, the division axis was not oriented (‘random division axis’). The thin clonal stripes suggested that NR stem cells had a preferential axis of division along the radial (central-peripheral) coordinate, while circumferential divisions occurred with lower frequency than expected for a random division axis orientation. We wondered whether NR stem cell division orientation could relate to shaping the organ. An inducer growth mode does not necessarily imply regulation of organ shape. To use an analogy, a mass of dough grows from within (similar to the inducer growth mode), but its shape can be imposed externally by a mold (i. e. the dough does not affect shape regulation). In the NR, the shape could plausibly be imposed externally by any of the surrounding tissues, and in this case, it would have no role in organ shape regulation (Figure 5A). As the space available for cells is imposed externally, any orientation of division axes is theoretically possible; after division cells will locally shift to optimally fill space. In an alternative scenario, organ shape could be regulated by oriented cell divisions of CMZ stem cells (Figure 5B’). In this scenario, a precise orientation of division axes is necessary. We calculated the ideal proportion of circumferential and radial divisions required to maintain hemispherical geometry. We assumed two principal axes of division, and that each new cell contributed either to the area of the CMZ or to the rest of the eye (Figure 5B’’). Circumferential divisions (two daughter cells stay in the CMZ) must be balanced by radial divisions (one daughter cell is poised to leave the niche and differentiate). A hemispherical eye of radius R has the area (1) Aeye=2πR2, while the CMZ forms a band of width w at the base of the eye with area (2) ACMZ=2πRw. Thus, we obtain an ideal ratio of circumferential to radial divisions of1: Aeye−ACMZACMZ, (3) 1: R−ww, that is for every one circumferential division, there must be R−ww radial divisions. Since R≫w, radial divisions must be more frequent than circumferential divisions, and the frequency of radial divisions increases as the retinal radius grows. To quantify circumferential stem cell divisions in experimental and simulated data, we took advantage of the exquisite temporal order of NR growth to measure ArCoS width – a proxy for circumferential stem cell divisions. To this end, we developed a pipeline that unrolled the retina as described before, and measured the number of pixels along each radial position normalized by the total circumference – effectively the angle enclosed by two rays traversing the center of the embryonic retina and the clone boundaries at every radial position (Figure 5D’’). To only include lifelong stem cells, we focused our analysis on the post-embryonic retina and excluded the central portion including the induction ring. As expected, with increasing probability to divide along the circumferential axis, average clone width increases in the simulation (Figure 5—figure supplement 1A’–B). When division axes perfectly match the ratio in Equation 3, the simulation becomes the limiting case of shape regulation where the hemispherical shape is always maintained. Thus, we modelled how the ‘ideal division axis’ ratio given by Equation 3 affected simulated ArCoS in the inducer growth mode and compared this to experimental data as well as simulations with random division axis (Figure 5C’–C’’’). Experimental ArCoS width averaged to 4. 87° (Figure 5D’ black graph; n = 99 ArCoS across seven retinae). In contrast to experimental data, ArCoS width in simulations with random division axis averaged to 7. 28° (Figure 5D’ blue graph; n = 102 clones from five simulation runs; compared to experimental data: p=1. 94∙10−7, Welch two-sample t-test). In simulations with ideal division axis, ArCoS width closely matched experimental data, averaging at 4. 54° (Figure 5D’, red graph; n = 133 clones from five simulation runs; compared to experimental data: p=0. 37, Welch two-sample t-test). These data show that NR stem cell divisions were not randomly oriented, but instead were preferentially oriented along the central-peripheral axis. Moreover NR stem cells underwent radial and circumferential divisions at a rate consistent with a role in organ shape regulation. We observed that in the retina of the surface-dwelling medaka, the position of the embryonic retina was not centered, but instead was shifted ventrally (Figure 6A’). As a result, the post-embryonic retina was longer dorsally than ventrally (ratio dorsal to ventral length: mean = 1. 42; standard deviation = 0. 29; n = 10 retinae). The embryonic retina covered the entire retinal surface at induction (Figure 6A’’). Equal growth around the circumference should maintain the embryonic retina in the center. The ventral-ward shift indicated that along the CMZ circumference, ventral stem cells had different division parameters. We probed the feasibility of different scenarios in generating a ventral shift in an in silico screen. First, we discerned two ways for stem cells in the ventral domain (defined as a 90° sector; Figure 6—figure supplement 2) to select a different division behavior: Either a lineage-bound intrinsic signal (e. g. epigenetic imprinting), or a lineage-independent extrinsic signal (e. g. a local diffusible molecule). Second, we altered two cell division parameters: The probability of division, which we varied between half (pdiv_ventral=0. 5⋅ pdiv_non-ventral) or equal to the value in the non-ventral sector (pdiv_ventral= pdiv_non-ventral), and the preferential axis of cell division, which we varied between circumferentially-biased (pcirc_ventral=1) and radially-biased (pcirc_ventral=0). In control simulations where all cells behaved equally, the embryonic retina stayed centered (Figure 6B’, C’). For a lineage-bound intrinsic signal, a circumferential bias lead to massive enlargement of ventral lineages at the expense of adjacent clones without affecting the embryonic retina (Figure 6B’’). Reducing proliferation probability resulted in termination of ventral lineages, as adjacent clones displaced them from the virtual niche (Figure 6B’’’). An intrinsic signal resulted in a ventral shift only if circumferential bias was combined with lower proliferation probability (Figure 6B’’’’ – condition I). In these simulations, circumferential divisions allowed ventral lineages to physically occupy niche positions (preventing their displacement) while lower proliferation reduced pressure on cells of the embryonic retina, allowing a ventral shift. In the scenario of a lineage-independent extrinsic signal, two conditions resulted in a ventral shift of the embryonic retina: Both lower division probability (Figure 6C’’’ – condition II) and the combination of lower division probability with circumferential division axis bias (Figure 6C’’’’ – condition III). To identify which scenario was most plausible, we analysed patches in the ventral and non-ventral sectors. Both in experiments and all three simulated conditions, patch shape in the non-ventral sector was similar (Figure 6D’–D’’’’). Although there was a tendency for ventral clones to terminate more often, the width distribution of experimental NR patches did not differ substantially between non-ventral and ventral sectors (Figure 6D’, E’, Figure 6—figure supplement 1D’; p=0. 84, Wilcoxon rank sum test). In contrast, this latter criterion was violated by two of the three simulated scenarios (Figure 6D’’–D’’’’ and E’’–E’’’’, Figure 6—figure supplement 1D’’-D’’’’). In condition I, ventral ArCoS started narrow but then broadened (Figure 6E’’) and interdigitated circumferentially (Figure 6—figure supplement 1A, black arrowheads), unlike the very uniform stripes in the experimental data. The broader ventral ArCoS lead to a more dispersed distribution compared to the non-ventral sector (Figure 6—figure supplement 1D’’; p=4. 31∙10−14, Wilcoxon rank sum test). In condition II, the majority of ventral ArCoS formed very narrow stripes, but at the border to the non-ventral sector ArCoS were broad and curved (Figure 6—figure supplement 1B, black arrowheads). Again, this resulted in more shape variation (Figure 6E’’’). Nevertheless, these outliers were outweighed by a high density of narrow clones, such that the overall distribution was similar between ventral and non-ventral sectors (Figure 6—figure supplement 1D’’’; p=0. 12, Wilcoxon rank sum test). Clones in the ventral and non-ventral sectors were qualitatively similar in condition III (Figure 6E’’’’, Figure 6—figure supplement 1C). Ventral clones however tended to be broader, resulting in a more dispersed distribution compared to the non-ventral sector (Figure 6—figure supplement 1D’’’’; p=7. 29∙10-7, Wilcoxon rank sum test). In conclusion, ventral NR stem cells have a different behavior than elsewhere along the circumference, leading to a ventral-ward shift of the embryonic retina. The simulations suggest that this different behavior consists of modulation of proliferation parameters by an extrinsic signal in the ventral CMZ. The coordinated growth of multiple independent tissues is a ubiquitous process in biology. In this work, we used the post-embryonic growth of NR and RPE in the eye of medaka as a model system of coordination in an organ where both growth and shape must be precisely regulated. Eye size in fish scales to the body size (Lyall, 1957; Johns and Easter, 1977). Body size, and thus eye growth rates greatly vary among individuals and depend on environmental factors (Johns, 1981). This natural malleability implies that feedback coupling plays a dominant role rather than the precise parametrization of each tissue growth and cell proliferation rate. Our simulations showed that inducer and responder growth modes impacted on variability in cell division timing, ultimately resulting in distinct clonal patterns that reproduced the experimentally observed differences between NR and RPE. RPE cells divided with high variability, indicative of periods of long quiescence where they waited for proliferative cues. NR cells displayed lower variability, supporting an upstream role in regulating growth (Figure 7A). Although our implementation of the responder growth mode used a mechanical stimulus (local cell density), a biochemical stimulus could equally well represent the system. Our model highlights an underappreciated mechanism whereby tissues coordinate by inducer and responder roles. Such division of labor among tissues might apply more generally to multiple organ systems, for example hair follicle cells in mouse induce the growth of underlying adipose tissue through hedgehog signalling (Zhang et al., 2016). Intriguingly, hedgehog signalling also regulates the NR/RPE boundary in the CMZ of medaka (Reinhardt et al., 2015), suggesting that signals mediating coordination of proliferative cell populations might be conserved. The topology of the retinal niche lead to a spatially biased neutral drift where stem and progenitor compartments spontaneously emerged. All virtual cells had equal potency, yet only a fraction realized their full stem cell potential. Peripheral cells had a high chance to become canalized in a stem cell fate, while central cells were more likely to act as progenitor cells with limited proliferation potential (Figure 7B). Our experimental data support a spatially biased neutral drift. Fusion of clones may have lead us to overestimate ArCoS deriving from the central domain, which represent progenitors reverting to a stem cell fate. Nevertheless, terminated clones arising from the very periphery of the niche unambiguously demonstrate that some stem cells failed to self-renew throughout the life of the animal. Moreover, our finding that only cells in the first two rows of the CMZ have stem cell potential is consistent with in vivo time-lapse data (Wan et al., 2016; Tang et al., 2017). Interestingly, retrograde movement of row 2 cells into row 1 of the CMZ occurs in vivo (Wan et al., 2016), which we also observed in our simulations. CMZ progenitor cells can be subdivided into two populations (Harris and Perron, 1998; Raymond et al., 2006): First, peripheral multipotent progenitors (i. e. able to generate all retinal neurons and glia) which differ from stem cells only in their proliferative potential. Second, central progenitors that are restricted both in proliferative and differentiation potential, which likely act as a transit-amplifying zone, both increasing the proliferative output and cross-regulating to produce a full neuronal complement with the correct proportions of cell types (Pérez Saturnino et al., 2018). Our data support an alternative model that identifies peripheral multipotent progenitors as stem cells that have been outcompeted. All terminated clones we examined were multipotent and spanned all retinal layers (Figure 1—figure supplement 2). Thus, as in many other systems (Clevers and Watt, 2018), our work highlights the limitation of strictly defining stem cells as infinitely self-renewing, or a posteriori based on their ArCoS-forming capacity. Importantly, although stochastic competition is most apparent in the early phase after clonal induction, it occurs continuously as demonstrated by late arising patches (Figure 3E) and nested inductions (Figure 4A’–A’’). The shift from an ‘early stochastic’ to ‘late polyclonal’ growth observed in other systems (Nguyen et al., 2017) may simply result from clonal growth masking the underlying stochasticity. Due to this stochasticity, it is impossible to tell at any moment with absolute certainty if a given cell will perpetually function as a stem cell. Neutral drift in a finite-sized environment such as adult mammalian tissues must ultimately result in a monoclonal niche (Snippert et al., 2010; Colom and Jones, 2016; Clevers and Watt, 2018). In fish, homeostatic growth expands niches, and thus the number of stem cells increases (Centanin et al., 2011). In principle, niche expansion reduces the impact of competition on clonal loss, but does not completely abolish it. Indeed, neutral drift leads to gradual loss of polyclonality in the intestine and muscle of fish (Aghaallaei et al., 2016; Nguyen et al., 2017). Organs may limit monoclonal drift by physically isolating niches (Aghaallaei et al., 2016). In the intestine of both mammals and fish, physical isolation of multiple niches results in a polyclonal organ built up of monoclonal units (Snippert et al., 2010; Aghaallaei et al., 2016). In contrast, the CMZ is a physically contiguous niche that nevertheless maintains polyclonality lifelong both in the NR and the RPE (Centanin et al., 2011; Centanin et al., 2014). As shown in this work, the retina is not devoid of stochastic competition. Then how does it conserve its polyclonality? Conceptually, the clonal growth of the retina resembles a population expanding into a new habitat, as studied in the context of evolutionary theory (Hallatschek and Nelson, 2010). Specifically for a radially expanding population, it has been mathematically proven that (assuming pure neutral genetic drift) no single clone will ever take over and clonal sectors perpetually coexist (Hallatschek and Nelson, 2010; Korolev et al., 2012). Growth of the perimeter is faster than circumferential expansion of clones, thus preserving population diversity (Hallatschek and Nelson, 2010). Interestingly, in the NR, the biased division axis further reduces competition (Figure 5), thus increasing niche polyclonality. In summary, the geometry of the CMZ niche prohibits the total loss of polyclonality. Our analysis of NR cell divisions implies that cells sense the radius of the eye to regulate organ shape. Across vertebrates, the retina integrates visual input to adapt organ shape to optimize optics, a process called ‘emmetropization’ (Wallman and Winawer, 2004). In chicken, emmetropization is regulated by specialized neurons distributed across the retina that send their axons to the CMZ, implicating the CMZ in regulation of eye shape (Fischer et al., 2008). Visual cues also guide emmetropization in fish (Kröger and Wagner, 1996; Shen et al., 2005; Shen and Sivak, 2007). Eye growth in young fish predominantly occurs by cell addition, while in older fish CMZ proliferation decreases (Johns, 1981) coincident with a decrease in emmetropization plasticity (Shen and Sivak, 2007). Thus, in fish, emmetropization correlates with CMZ proliferation. Experiments in chicken and zebrafish support the existence of two principal axes of stem cell division, that is circumferential and central-peripheral (Fischer et al., 2008; Ritchey et al., 2012; Wan et al., 2016). Notably, the predominance of central-peripheral divisions and decreasing frequency over time of circumferential divisions in CMZ stem cells that is predicted by Equation 3 is supported by in vivo imaging data (Wan et al., 2016) and previous long-term clonal analyses (Centanin et al., 2014). Altogether, the data support a model where the NR perceives the retinal radius through visual cues, and that cell divisions in the NR contribute to shaping the eye. The retinae of many fishes grow asymmetrically, perhaps to maintain the relative positions of receptive fields of neurons (Johns, 1977; Johns, 1981; Easter, 1992). Ecology dictates a distribution of subdomains enriched in specialized neuronal circuits and retinal cell subtypes (Zimmermann et al., 2018). Interestingly, in green sunfish, the area that grows slowest displays highest visual acuity (Cameron, 1995). Medaka predominantly gaze upwards in their native shallow rice paddies, and a higher ventral acuity has been presumed based on photoreceptor densities (Nishiwaki et al., 1997). Thus, slower ventral growth may have evolved to match ecological requirements for medaka vision. Our in silico screen identified three scenarios consistent with asymmetric ventral growth. Based on clonal patterns, an extrinsic signal driving lower proliferation (and potentially also circumferential divisions) appears most plausible. Experimental eye re-orientation in vivo implied an eye-internal mechanism independent on body axes or visual cues in regulating asymmetric retinal growth (Cameron, 1996). The origin of this signal and how it scales with the growing eye to always affect a similarly-sized retinal sector remains to be elucidated. The retina integrates global systemic cues such as nutrition to scale with body size (Johns and Easter, 1977), local eye-internal cues to generate an asymmetric retinal topology (Cameron, 1996), and external visual cues to adapt the shape of the organ (Kröger and Wagner, 1996; Shen and Sivak, 2007). In chicken and goldfish, visual cues and nutrients feed into the CMZ through growth factor signalling (Boucher and Hitchcock, 1998; Fischer et al., 2008; Ritchey et al., 2012). We propose that NR cells in the CMZ act as a hub to coordinate organ growth; in the eye of fish, this happens at the level of cell proliferation parameters, which affect eye growth, eye shape, and retinal topology (Figure 7C). Indeterminate, lifelong growth is a widespread evolutionary strategy (Karkach, 2006). Given the geometrical constraints of the eye with respect to optics, a peripheral proliferative domain is the most parsimonious architecture to ensure that the differentiated neuronal cell mosaic is not disturbed by constant proliferation. Fishes are the largest vertebrate clade, with a huge diversity of eye shapes, such as cylindrical eyes in deep-sea fish (Fernald, 1990). By modulating CMZ proliferation parameters, evolution can adapt whole-organ morphogenesis to perfectly fit to the species’ ecological niche. | Title: Retinal stem cells modulate proliferative parameters to coordinate post-embryonic morphogenesis in the eye of fish Summary: By the time babies reach adulthood, they have grown many times larger than they were at birth. This development is driven by an increase in the number and size of cells in the body. In particular, special types of cells, called stem cells, act as a reservoir for tissues: they divide to create new cells that will mature into various specialized structures. The retina is the light-sensitive part of the eye. It consists of the neural retina, a tissue that contains light-detecting cells, which is supported by the retinal pigment epithelium or RPE. In fish, the RPE and neural retina are replenished by distinct groups of stem cells that do not mix, despite the tissues being close together. Unlike humans, fish grow throughout adulthood, and their eyes must then keep pace with the body. This means that the different tissues in the retina must somehow coordinate to expand at the same rate: otherwise, the retina would get wrinkled and not work properly. Tsingos et al. therefore wanted to determine how stem cells in the neural retina and RPE co-operated to produce the right number of new cells at the right time. First, stem cells in the eyes of newly hatched fish were labelled with a visible marker so that their divisions could be tracked over time to build cell family trees. This showed that stem cells behaved differently in the neural retina and the RPE. Computer simulations of the growing retina explained this behavior: stem cells in the neural retina were telling the RPE stem cells when it was time to divide. Combining results from the simulations with data from the experiments revealed that a stem cell decided to keep up dividing partly because of its position in the tissue, and partly because of random chance. To be healthy, the body needs to fine-tune the number of cells it produces: creating too few cells may make it difficult to heal after injury, but making too many could lead to diseases such as cancer. Understanding how tissues normally agree to grow together could therefore open new avenues of treatment for these conditions. | 12,478 | 462 | lay_elife | en |
Summarize: Leonardo DiCaprio has been the first to admit that he went to some extreme lengths while filming The Revenant—the wilderness epic that may finally earn the four-time nominated actor a deserved Oscar. To play a 19th-century frontiersman seeking revenge on Tom Hardy’s trapper, for example, DiCaprio slept in animal carcasses; submerged himself in frozen rivers, consistantly risked hypothermia during the icy, outdoors shoot; and ate raw bison liver. One activity in which DiCaprio definitely did not partake, however, was a bear rape scene, as the Drudge Report claims on Tuesday in typically tasteful headline fashion: “DICAPRIO RAPED BY BEAR IN FOX MOVIE.” We know DiCaprio is not raped by a bear in The Revenant because, dear readers, there is simply no such scene in the admittedly grisly Alejandro González Iñárritu-directed film. Granted, DiCaprio does face-off with a bear in a showdown so violent it might deter you from the grizzly exhibit the next time you visit the zoo. But the actor is not sexually assaulted by a bear once, let alone twice, as the right-wing news outlet alleges in its bonkers story. Pull up a chair and, to correctly express the outraged incredulousness implied by the report’s punctuation, a megaphone. The new movie 'REVENANT' features a shocking scene of a wild bear raping Leo DiCaprio! The explicit moment from Oscar winning director Alejandro Inarritu has caused maximum controversy in early screenings. Some in the audience escaped to the exits when the Wolf of Wall Street met the Grizzly of Yellowstone. The story of rural survivalism and revenge reaches new violent levels for a mainstream film. The bear flips Leo over and thrusts and thrusts during the explicit mauling. "He is raped -- twice!" Not to be outdone, DiCaprio rips open a horse and sleeps naked in its carcass! DiCaprio does indeed tango violently with a bear, with the grizzly flipping the actor over and attacking his back. As if the lack of inter-species molestation is enough to disprove Drudge's report, several co-workers also point out that the film suggests that the bear attacker is female. DiCaprio's character is attacked after he makes the mistake of trespassing on bear cub turf. (“Is there such a thing as an overly-protective single father bear?” a colleague rightly inquired.) In an interview this past October, the DiCaprio discussed filming the attack scene, which involved cables and precise choreography. “[Those scenes]—amongst many other sequences—were some of the more difficult things I’ve ever had to do in my entire career,” DiCaprio told Yahoo!. “But the end result is going to be one of the most immersive experiences audiences will ever have with what it would be like to come face-to-face with an animal of that magnitude that is incredibly primal.” The ridiculous report seems to have stemmed from veteran Hollywood journalist Roger Friedman, who reviewed the film on his blog Showbiz411 on November 29, and has a very different recollection of the bear attack scene than his peers. “The bear flips [DiCaprio’s character] over on his belly and molests him– dry humps him actually– as he nearly devours him,” Friedman writes, adding that he did not entirely see the sequence as a negative. In fact, he is impressed by the technicality involved. “How Innaritu and DiCaprio did this is a movie mystery because it is as real feeling as Bruce the shark in Jaws 40 years ago. It’s as real looking as it could be, and maybe the most frightening moment I’ve seen in a film in eons.” Although Drudge’s bear-rape alarm may not be factual, it has certainly been enjoyably ridiculous by many a Twitter user, who have spent their morning conjuring up clever digs at the conservative blog. But perhaps the most sound reaction to DiCaprio Bear Rape Gate—and the best indicator of how overblown this report became in such a small time—is this very good point from Jeb Bush's communications director Tim Miller. (Yes, that Jeb!) Drudge conspiracy theorists, feel free to take a look at the bear fight footage in the trailer below. And lastly, our thoughts go out to the media maligned bear of The Revenant, who is likely hiring a publicist, lawyer-ing up, and beginning the early stages of her defamation suit this very minute. The Revenant opens in theaters on January 8. Get Vanity Fair’s HWD Newsletter Sign up for essential industry and award news from Hollywood. E-mail Address Subscribe Advance screenings of Alejandro Gonzalez Innaritu’s epic “The Revenant,” starring Leonardo DiCaprio, are producing all kinds of reactions, all wildly favorable, albeit with caveats. The Fox movie, based on Michael Punke’s “novel of revenge,” takes place near Yellowstone, Montana, in 1823. It begins with the same bloody incident that launches the book– the gruesome attack by a grizzly bear on trapper Hugh Glass. Innaritu has taken essentially the following sections of Punke’s book and enlarged them into a feasting by animal on man. The bear flips Glass over on his belly and molests him– dry humps him actually– as he nearly devours him. How Innaritu and DiCaprio did this is a movie mystery because it is as real feeling as Bruce the shark in “Jaws” 40 years ago. It’s as real looking as it could be, and maybe the most frightening moment I’ve seen in a film in eons. You can see a little bit of the frightening bear attack in this trailer: Here’s the passage from Punke’s book. Nothing is spared in the filming: First the bear attacks Glass: The grizzly dropped to all fours and was on him. Glass rolled into a ball, desperate to protect his face and chest. She bit into the back of his neck and lifted him off the ground, shaking him so hard that Glass wondered if his spine might snap. He felt the crunch of her teeth striking the bone of his shoulder blade. Claws raked repeatedly through the flesh of his back and scalp. He screamed in agony. She dropped him, then sank her teeth deep into his thigh and shook him again, lifting him and throwing him to the ground with such force that he lay stunned— conscious, but unable to resist any further. He lay on his back staring up. … Then Glass is found after the attack by Harris, one of the trappers: …he had never seen human carnage like this, fresh in the wake of attack. Glass was shredded from head to foot. His scalp lay dangling to one side, and it took Harris an instant to recognize the components that made up his face. Worst was his throat. The grizzly’s claws had cut three deep and distinct tracks, beginning at the shoulder and passing straight across his neck. Another inch and the claws would have severed Glass’s jugular. As it was, they had laid open his throat, slicing through muscle and exposing his gullet. The claws had also cut the trachea, and Harris watched, horrified, as a large bubble formed in the blood that seeped from the wound. It was the first clear sign that Glass was alive. Harris rolled Glass gently on his side to inspect his back. Nothing remained of his cotton shirt. Blood oozed from deep puncture wounds at his neck and shoulder. His right arm flopped unnaturally. From the middle of his back to his waist, the bear’s raking claws left deep, parallel cuts. It reminded Harris of tree trunks he had seen where bears mark their territory, only these marks were etched in flesh instead of wood. On the back of Glass’s thigh, blood seeped through his buckskin breeches. Harris had no idea where to begin…. It’s not the only shocking moment in “The Revenant.” Later in the movie– and the novel– Glass (DiCaprio) comes upon a dead horse, removes its insides, takes off his clothes and climbs inside the carcass for warmth during a storm. If you’ve already survived the bear scene, this is the coup de grace. And what will Fox do with two potential Best Picture nominees with lead actors and directors in direct competition? They already have a huge box office crowd pleaser with an Oscar performance by Matt Damon in “The Martian.” Every studio should have these problems! They’re almost un-bearable! read more of today’s headlines click here On Tuesday, the Internet exploded following a report that Oscar-winning director Alejandro González Iñárritu included a sequence in his new film, The Revenant, where Leonardo DiCaprio’s character is raped by a bear. Not so fast, says the film’s studio. “As anyone who has seen the movie can attest, the bear in the film is a female who attacks Hugh Glass because she feels he might be threatening her cubs,” a Fox spokesperson told Entertainment Weekly in an exclusive statement. “There is clearly no rape scene with a bear.” In The Revenant, which Fox will debut in limited release on Christmas Day, DiCaprio’s character is a frontiersman who confronts the bear and her cubs while out in the woods. The bear attack has been featured heavily in the film’s marketing campaign, and can be spotted in the trailer below at the 53-second mark. Earlier on Tuesday, the bear in The Revenant was nominated for Outstanding Achievement, Character Animation in a Live Action Production. The animal, named Judy, is up against Indominus Rex from Jurassic World, The Hulk from Avengers: Age of Ultron, and Azog and Smaug from The Hobbit: The Battle of the Five Armies in the category. Following its Christmas release, The Revenant will expand to nationwide theaters on Jan. 8. Fox would like everyone to know that Leonardo DiCaprio’s character does not get raped by a bear in “The Revenant.” On Tuesday, a report hit Matt Drudge’s Drudge Report that sent the Internet into a frenzy, claiming that DiCaprio’s character in the upcoming movie is victim to a violent rape at the hands of a bear. Journalists and other industry figures who have seen the movie subsequently clarified that DiCaprio is not raped, but simply brutally mauled by the bear. Fox, the studio behind the film, decided to also set the record straight by issuing a statement later on Tuesday, maintaining that the Drudge report is incorrect. “As anyone who has seen the movie can attest, the bear in the film is a female who attacks Hugh Glass because she feels he might be threatening her cubs,” a Fox spokesperson said in a statement to Entertainment Weekly. “There is clearly no rape scene with a bear.” The report on Drudge quotes a source saying that “He is raped – twice!,” and claims that people in the audience walked out of theaters in disgust of the scene. “The Revenant,” directed by Alejandro González Iñárritu and also starring Tom Hardy, debuts in limited release on Christmas Day, and goes wide Jan. 8. | Summary: The new Leonardo DiCaprio movie The Revenant features no scenes whatsoever of the star being raped by a bear, Fox stressed after bizarre rumors swept the Internet on Tuesday. The studio decided it had to clarify the issue after a Drudge Report story claimed that DiCaprio's character, frontiersman Hugh Glass, was raped twice as part of a brutal mauling, Variety reports. "As anyone who has seen the movie can attest, the bear in the film is a female who attacks Hugh Glass because she feels he might be threatening her cubs," a Fox rep tells Entertainment Weekly. "There is clearly no rape scene with a bear." Vanity Fair traced the rumor to Hollywood journalist Roger Friedman, who, unlike every other reviewer, interpreted the mauling as molestation. In a Showbiz411 post, Friedman declared the scene with a CGI bear "a movie mystery because it is as real feeling as Bruce the shark in Jaws 40 years ago." "It's as real looking as it could be, and maybe the most frightening moment I've seen in a film in eons," he wrote. Vanity Fair notes that even Jeb Bush's communications director joined the debate over the scene, tweeting: "Are there any recorded examples of a bear raping a human in real life?" | 2,560 | 288 | multi_news | en |
Write a title and summarize: V1 and V2b interneurons (INs) are essential for the production of an alternating flexor–extensor motor output. Using a tripartite genetic system to selectively ablate either V1 or V2b INs in the caudal spinal cord and assess their specific functions in awake behaving animals, we find that V1 and V2b INs function in an opposing manner to control flexor–extensor-driven movements. Ablation of V1 INs results in limb hyperflexion, suggesting that V1 IN-derived inhibition is needed for proper extension movements of the limb. The loss of V2b INs results in hindlimb hyperextension and a delay in the transition from stance phase to swing phase, demonstrating V2b INs are required for the timely initiation and execution of limb flexion movements. Our findings also reveal a bias in the innervation of flexor- and extensor-related motor neurons by V1 and V2b INs that likely contributes to their differential actions on flexion–extension movements. Terrestrial animals use their limbs to generate a broad array of motor behaviors, from stereotypical movements that include protective reflexes and locomotion to complex volitional tasks that are exemplified by reaching and grasping movements (Grillner, 1975; Alstermark and Isa, 2012). Charles Sherrington (1906) first demonstrated that all such motor behaviors rely on the reciprocal actions of flexor and extensor muscles around each limb joint and that terrestrial animals require reciprocal inhibition to move and articulate their limbs. It is known that reciprocal flexor–extensor motor activity is produced by inhibitory neurons in the spinal cord, many of which appear to be core components of the locomotor central pattern generator (CPG) (reviewed in Kiehn, 2006; Goulding, 2009; Grillner and Jessell, 2009; Arber, 2012). However, the functional organization of the inhibitory circuits that control flexor–extensor activity and the contribution that different inhibitory neuron cell types make to flexor–extensor motor control is still poorly understood. Studies in the cat have identified a number of physiologically defined IN cell types that are candidates for exercising flexor–extensor control. The most prominent of these are reciprocal Ia inhibitory interneurons (IaINs), which are activated by muscle spindle afferents and inhibit antagonist motor neurons. IaINs are rhythmically active during locomotion (Feldman and Orlovsky, 1975; Pratt and Jordan, 1987) and scratching (Deliagina and Orlovsky, 1980). Peak IaIN activity coincides with the phase in which antagonist motor neurons are hyperpolarized (Pratt and Jordon, 1987; Geertsen et al., 2011). This activity profile is strong evidence of a central role in reciprocal inhibition. The contribution that non-reciprocal inhibitory Ib interneurons (IbINs) make to locomotion is less clear. While the IbINs that are innervated by Golgi tendon organs (GTOs) generally inhibit homonymous motor neurons under non-locomotor conditions (Jankowska, 1992; Pearson and Collins, 1993), during locomotion the Ib pathway exerts an excitatory effect on extensor motor activity (Conway et al., 1987; Pearson and Collins, 1993; Gossard et al., 1994; Angel et al., 2005). Nonetheless, inhibition mediated by IbINs has been observed during the extension phase of walking in humans (Shoji et al., 2005). This inhibition is associated with the unloading of the limb, and it suggests that IbINs may contribute to the phase transition from stance (extension) to swing (flexion). The role that Renshaw cells play in shaping flexor–extensor locomotor activity appears to be more limited (Pratt and Jordan, 1987). While these cells are rhythmically active during locomotor activity, reducing Renshaw cell transmission by pharmacological or genetic blockade of cholinergic transmission changes the periodicity of the locomotor rhythm with little discernible effect on flexor–extensor alternation (Noga et al., 1987; Myers et al., 2005). Genetic analyses in mice have identified a number of molecularly defined interneuron populations that provide inhibition to motor neurons (reviewed in Goulding, 2009; Arber, 2012). Among these are three prominent populations of ipsilaterally projecting inhibitory interneurons: dorsal Lbx1-derived inhibitory INs (Gross et al., 2002; Tripodi et al., 2011) and ventral V1 and V2b INs (Saueressig et al., 1999; Sapir et al., 2004; Zhang et al., 2014). The V1 and V2b IN populations are both heterogeneous, being made up of multiple physiological cell types including Renshaw cells (V1 INs), IaINs (V1 and V2b INs) and putative IbINs (V2b INs) (Sapir et al., 2004; Alvarez et al., 2005; Zhang et al., 2014), as well as other as yet unidentified inhibitory cell types. Recently, we have found that the composite activities of the V1 and V2b INs are required to secure flexor–extensor alternation in the in vitro neonatal spinal cord and in newborn mice (Zhang et al., 2014). This finding is consistent with our demonstration that cells with the features of IaINs are derived from both V1 and V2b INs and that disynaptic reciprocal inhibition is only abolished when both of these populations are functionally inactivated (Wang et al., 2008; Zhang et al., 2014). The limited repertoire of motor behaviors that can be assayed using the in vitro neonatal spinal cord preparation, together with our inability to discern any marked differences in the function of V1 and V2b INs with respect to generating an alternating flexor–extensor motor rhythm in vitro, prompted us to examine the contribution that V1 and V2b INs make to motor control in awake behaving mice. In particular, we were interested in determining whether these two inhibitory interneuron classes control discrete aspects of limb movement with regard to flexor–extensor-driven motor behaviors. Our results demonstrate a striking functional bias in the actions of V1 and V2b INs on flexor–extensor motor activity, whereby the selective ablation of V1 vs V2b INs results in hindlimb hyperflexion and hyperextension, respectively. This functional bias was observed both in air-stepping juvenile mice and in adult animals performing motor tasks such as over-ground walking. In analyzing the inhibitory inputs to motor neurons from these two interneuron populations, we find a genetically defined bias in V1 and V2b connectivity that likely underpins the differential effects on flexor–extensor motor activity. Specifically, a higher proportion of the inhibitory contacts on flexor motor neurons come from V1 INs as compared to extensor motor neurons, whereas V2b INs preferentially contact motor neurons that innervate extensor muscles. To determine the efficiency and specificity of this technique, we monitored V1 IN ablation in P1 En1Cre; Cdx2-FlpO; Maptds-DTR mice treated with a single dose of DTX and analyzed 6 days later. P1 control mice (En1Cre; Maptds-DTR) were also treated with DTX and analyzed. Because En1 is no longer expressed postnatally, an Ai6 Cre-dependent reporter allele (Madisen et al., 2010) was used to independently mark the V1 INs and verify their loss following DTX treatment. Whereas V1 INs were largely spared at upper cervical levels (Figure 2A, B), they were depleted by >90% in the thoracic, lumbar, and sacral spinal cord of En1Cre; Cdx2-FlpO; Maptds-DTR; Ai6 mice (Figure 2C, D). Calbindin+ Renshaw cells, which are derived from En1+ progenitors (Sapir et al., 2004), were also largely missing from the caudal spinal cord (Figure 2E, F). By contrast, dorsal calbindin+ neurons that are not derived from En1+ progenitors were still present in normal numbers (Figure 2—figure supplement 1). 10. 7554/eLife. 04718. 004Figure 2. Restricted ablation of V1 and V2b INs following diphtheria toxin treatment. Immunohistochemical and histological analysis of P7 control, En1Cre; Cdx2-FlpO; Maptds-DTR and Gata3Cre; Cdx2-FlpO; Maptds-DTR animals 6 days after administering DTX. All sections are from the mid-lumbar cord except for those in panels A, B, M, and N, which are from the cervical cord. (A–D) En1-derived V1 INs (green) are selectively ablated in the lumbar spinal cord (c. f. C, D), whereas ChAT+ motor neurons (red) are spared at lumbar (D) and cervical levels (B). (E, H) Calbindin+ Renshaw cells (blue) are present in control cords (E, arrow) but not in V1 IN-ablated cords (F). (G, H) Chx10+ V2a INs are present in normal numbers following V1 IN-ablation. (I, J) Hematoxylin-eosin staining reveals no evidence of widespread neuronal cell loss or gliosis. (K, L) CD86+ microglia were occasionally observed in close proximity to V1 cell debris (arrowhead). (M–P) Motor neurons are spared at both cervical (M, N) and lumbar (O, P) levels following ablation of the V2b INs. (Q, R) V2b INs (green) are specifically deleted in V2b IN-ablated mice (R) while V0c neurons (red) are spared. (S, T) Chx10+ V2a INs are present in normal numbers following V2b IN-ablation. (U, V) Hematoxylin-eosin staining. (W, X) Localized CD86 expression (red) in lamina VII is associated with dying V2b INs (green). DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 00410. 7554/eLife. 04718. 005Figure 2—figure supplement 1. Selective ablation of ventral calbindin-expressing neurons. (A, B) Expression of calbindin in the dorsal spinal cord of P7 control (A) and V1 IN-ablated mice (B). (C) V1 IN cell numbers per hemisection at thoracic and lumbar levels (n = 15 sections [30 μm] from 3 spinal cords each for control and V1 IN-ablated mice). (D, E) Counts of neurons in the lumbar spinal cord of P7 DTX-treated mice (6 days p. i.). Cell counts at the indicated levels were expressed as a percentage of comparable counts for control sections (n = 12 sections [30 μm] for control), V1 IN-ablated and V2b IN-ablated mice (n = 3 cords for each genotype). Data are expressed as mean ± s. d. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 005 To confirm the specificity of V1 IN cell killing, sections from control and V1 IN-ablated cords were stained with an antibody to choline acetyltransferase (ChAT) to visualize motor neurons and cholinergic V0c INs. Both populations were unaffected by the DTR-dependent ablation of V1 INs (Figure 2A–D, data not shown). Moreover, there was no reduction in the number of Chx10+ V2a INs, a prominent population of excitatory neurons that are intermingled with V1 INs in lamina VII (Figure 2G, H). Further histological analysis revealed no change in the integrity of the spinal cord following V1 IN ablation (Figure 2I, J) nor was there any reduction in neuronal cell numbers (Figure 2—figure supplement 1D). We did observe a small transient increase in CD86 expression as is expected with the targeted killing of V1 INs (Figure 2K, L). This CD86 expression was localized to lamina VII where V1 INs reside, and in most instances co-localized with GFP-labeled V1 cell debris (Figure 2L, arrowhead). Most importantly, CD86 expression was not widespread nor was there any evidence of infiltration by CD45R-positive B cells or CD3-positive T cells (data not shown). These results agree with other studies showing DTR-mediated cell killing is highly selective and cell autonomous (Buch et al., 2005; Hatori et al., 2008). A similar analysis was performed on Gata3Cre; Cdx2-FlpO; Maptds-DTR mice post DTX-treatment (Figure 2M–X, Figure 2—figure supplement 1E). These mice also displayed a selective loss of V2b INs in the thoracic and lumbosacral spinal cord (Figure 2R). Other spinal neurons including motor neurons, V0c INs and Chx10+ V2a INs were completely spared in these cords (Figure 2M–T). Once again, we observed limited and localized expression of CD86 in lamina VII (Figure 2X). These data demonstrate that the intersectional ablation approach selectively and efficiently deletes V1 and V2b INs in caudal regions of the spinal cord. When DTX was administered to postnatal En1Cre; Cdx2-FlpO; Maptds-DTR pups, it produced a strong hyperflexion phenotype within 3–4 days (Figure 3). Whereas control P7 pups flexed and extended their hindlimbs when suspended by their tails (Figure 3A), the hindlimbs of P7 DTX-treated En1Cre; Cdx2-FlpO; Maptds-DTR V1 IN-ablated pups remained flexed (Figure 3B), even though their forelimbs were able to extend and display a full range of stepping movements (Figure 3B, asterisk). In contrast to control mice and V1 IN-ablated mice, P7 Gata3Cre; Cdx2-FlpO; Maptds-DTR V2b IN-ablated mice maintained their hindlimbs in an extended state when suspended by their tail (Figure 3C), even though they were still able to flex and extend their forelimbs. 10. 7554/eLife. 04718. 006Figure 3. Mice lacking V1 and V2b INs show abnormal hindlimb movements. Time-lapse sequence images showing the hindlimb movements of P7 mice suspended by their tails. Mice were photographed 4 days after DTX treatment. The genotypes of the mice are indicated. Control mice were littermates that lacked the Cdx2-FlpO allele. (A) Control mice are able to flex (arrowheads) and extend (arrow) their hindlimbs when suspended by their tail. (B) Following the ablation of V1 INs, P7 mice lose their ability to extend their hindlimbs, which remain clasped to the body in a flexed position (arrowheads). The forelimbs of these mice are able to undergo extension movements (asterisk). (C) Ablation of V2b INs leads to pronounced extension of the hindlimbs (arrows) and impairment of hindlimb flexion movements. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 006 We then analyzed the hindlimb locomotor movements of DTX-treated mice that were induced to airstep by subcutaneous injection with L-DOPA. Kinematic analysis revealed strong rhythmic stepping in both the forelimbs and hindlimbs of control animals (Figure 4). By contrast, V1 IN-ablated animals displayed very little hindlimb movement, in contrast to the forelimbs that showed a normal range of motion. The hindlimbs of V1 IN-ablated animals, while occasionally displaying small rhythmic twitches (Figure 4B, asterisks), remained flexed during stepping (see Figure 4A, middle panel, arrowheads). Moreover, the maximal opening of the ankle joint in these animals was less than 80° compared to 145° for control animals (Figure 4B, C). In those instances where ankle joint did partially open, the overall change in angle was reduced from 90° to less than 30° (Figure 4C). 10. 7554/eLife. 04718. 007Figure 4. Juvenile mice lacking V1 and V2b INs display abnormal hindlimb movements. (A) Images of airstepping P7 animals following DTX injection. Flexed limbs are depicted with black arrowheads and extended limbs with white arrows. Control animals (left) flex and extend their hindlimbs. The hindlimbs of V1 IN-ablated animals (middle) remain flexed and those of V2b IN-ablated mice (right) are fully extended. (B) Traces showing the change in ankle (top) and elbow (bottom) joint angle during 5 s of airstepping. Deletion of V1 and V2b INs impairs movement of the ankle joint, whereas rhythmic movements are maintained for the elbow joint. Small rhythmic variations of the ankle joint angle occur in phase with movements of the elbow joint angle (asterisks). (C) Measured changes in joint movement showing the amplitude change (left), the maximal joint angle (filled symbol) and the minimal joint angle (open symbol) joint angle (right). The mean and s. d. is shown for 24 consecutive steps (n = 3 animals for each genotype). DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 007 When V2b IN-ablated mice were induced to airwalk and analyzed in a similar manner, they displayed robust rhythmic forelimb stepping movements (Figure 4, right panels). By contrast, the hindlimbs of these mice remained extended and displayed very little in the way of flexion movements. Interestingly, L-DOPA also induced small rhythmic deflections of the hindlimb in these mice (Figure 4B, right panel, asterisk), indicating that the hindlimb locomotor CPG still produces an underlying locomotor rhythm. However, as the angle of the ankle joint was not reduced below 130° (Figure 4C), it appears that the hindlimb locomotor CPG is unable to elicit strong flexion movements in the absence of V2b inhibition. To delve more deeply into the nature of the motor deficits that underlie the opposing motor phenotypes that arise when V1 and V2b INs are ablated, fine EMG recording electrodes were implanted unilaterally in the tibialis anterior (TA) and gastrocnemius (GS) muscles to measure ankle flexor and extensor motor activity during L-DOPA-induced air-stepping. Control mice displayed a regular alternating pattern of EMG activity in both muscles (Figure 5A) in which there was little or no overlap in TA and GS burst activity. This profile of TA-flexor and GS-extensor activity in control P7 mice is very similar to that seen in adult mice (see also Figure 6), where longer duration GS bursts are interspersed with short TA bursts. The GS extensor phase was also seen to increase proportionately with longer step periods, while the TA flexor burst period was constant across all stepping speeds (Figure 5A, D). This is in strong agreement with previous studies performed in rodents and cats (Grillner, 1975; Halbertsma, 1983; Juvin et al., 2007; Frigon and Gossard, 2009) showing that changes in duration of the extensor/stance phase are preferentially responsible for lengthening or shortening the step cycle. 10. 7554/eLife. 04718. 008Figure 5. Altered rhythmic muscle activities during airstepping in the absence of V1 or V2b cells. (A) EMG recordings from the tibialis anterior (TA, flexor) and gastrocnemius (GS, extensor) muscles during L-DOPA-induced airstepping. Single deleted bursts are indicated by arrowheads. The asterisk marks a prolonged deletion that encompasses two step cycles. (B) Scatter plots show the relationship between ankle flexor (TA) burst duration and the step cycle period (black dots) and between ankle extensor (GS) burst duration and the step cycle period (gray circles). Each point represents the ratio of burst to step cycle duration for a single step. Control and V2b IN-ablated animals display an extensor dominant pattern, whereas V1 IN-ablated animals show a flexor-phase dominant pattern. Mean slope of regression calculations for each experimental group shows that the step cycle period in control mice (aGS = 0. 72) is strongly correlated with GS burst duration, while TA bursts remain relatively constant (aTA = 0. 12). The increase in step cycle duration in V1 IN-ablated mice is moderately correlated with flexor phase duration (aTA = 0. 46). V2b IN-ablated animals display a pronounced increase in step cycle duration that is very highly correlated with the length of the extensor phase (aGS = 0. 96). (C) Quantification of step cycle period, flexor and extensor burst duration for control, V1 IN-ablated and V2b IN-ablated mice. (D) Percentage of skipped bursts/deletions as measured during twelve 10-s periods of locomotor activity (n = 3 animals each). DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 00810. 7554/eLife. 04718. 009Figure 6. Mice lacking V1 and V2b INs have opposing changes to their gait. Hindlimb kinematics from control (left), V1 IN-ablated (middle), and V2b IN-ablated (right) mice at 3 weeks post DTX injection, by which time functional recovery was maximal. (A) Representative stick figure diagrams showing one complete step cycle (swing and stance) for the hindlimb. The arrow (middle) indicates hyperflexion during early swing phase in V1 IN-ablated mice. The arrowhead shows hyperextension of the ankle joint during late stance in V2b IN-ablated mice. (B) Comparison of limb positions at the transition from stance to swing (left), at mid-swing (middle), and at the swing to stance transition (right). (C) Representative angular changes to the ankle joint. The arrows indicate when the foot is lifted (stance to swing) and when the foot is planted (swing to stance). (D, E) Simultaneous EMG recordings of the GS and TA muscles in one leg during walking (D) and swimming (E). The bar in D indicates the expansion in TA activity. The asterisks indicate co-activation of the TA and GS muscles. Note the synchronous activity of both muscles during swimming in V1 IN-ablated mice. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 00910. 7554/eLife. 04718. 010Figure 6—figure supplement 1. Phase relationship between TA and GS EMG activity. The average period that the TA and GS muscles are active during the step cycle was calculated. Measurements were normalized for each step cycle, with the onset of GS EMG activity (0) serving as the reference for the beginning of the step cycle and 1 indicating the onset of the following stance phase. Note the expansion of TA activity in mice lacking V1 INs. Mice lacking V2b INs show prolonged GS activity during swing when the TA muscle is active. During swimming V1 IN-ablated mice show extensive co-activation of the GS and TA muscles. The mean period of muscle activity (gray solid bars) for each experimental sample is shown (n = 18 steps). Black error bars indicate the standard deviation for each sample. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 01010. 7554/eLife. 04718. 011Figure 6—figure supplement 2. Progressive changes in EMG activity following V1 IN ablation. Representative EMG recordings and kinematics from control (A), and V1 IN-ablated mice 5 (B) and 7 (C) days after commencing DTX treatment. The EMG recordings (upper panels) and joint angle plots (lower panel) are synchronized. In control animals, the TA and IP muscles are inactive during stance, and TA and GS activity is strictly alternating (A). The arrow in B indicates a small burst of ectopic TA activity which is associated with hyperflexion of the limb in V1 IN-ablated mice. By day 7 p. i., TA muscle activity has expanded and is co-active with the GS muscle during stance (C, bar). IP burst activity during swing (B, bar) and stance (arrowhead) is also increased. The matching lower traces show the accompanying changes in the angle of the hip, knee, and ankle joints during walking. Note the reduced opening of the hip and knee joints that accompanies the loss of V1 INs. At day 5 p. i., the ankle joint opens up more slowly (black arrows), but is still able to fully open. By day 7 p. i. the ankle joint is flexed throughout the step cycle, both at early stance (black arrow) and at the transition from stance to swing (asterisk). The small increase in hip motion seen at day 7 p. i. compared to day 5 p. i. is due to increased swaying of the pelvis during walking. The knee angle is also reduced during the step cycle. The shaded boxes indicate the extensor phase. Following V1 IN cell loss, ectopic TA muscle EMG activity is observed during stance (arrow). DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 01110. 7554/eLife. 04718. 012Figure 6—figure supplement 3. Altered limb and body movements in mice lacking V1 INs. (A) Foot placement analysis in control and V1 IN-ablated mice 3 weeks post-DTX treatment. High-speed video was used to capture the foot strike during walking on an elevated walkway. (Left) The position of the forepaws during stance is shown in blue, the position of the hindpaws are shown in red. The arrowhead indicates the first forelimb step, with the animals moving from right to left. (Right) The stride lengths for the forelimb and hindlimb were calculated for 36 steps (n = 3 animals each for control and V1 IN-ablated). (B) V1 IN-ablated mice show an increase in the rotation of the pelvis during walking. (Left) The change in the angle of the pelvis was calculated with 90° representing the median position of the pelvis. (Right) Quantification of the angular change in pelvis during a single step. The angular change was calculated for 24 steps (n = 3 animals each for control and V1 IN-ablated). Data is expressed as mean ± s. d. ** indicates p < 0. 01. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 012 V1 IN-ablated mice consistently displayed a marked increase in the duration of the TA burst, with the TA muscle remaining active for a proportionately longer period of each step cycle as compared to the GS muscle (Figure 5B). This finding is consistent with the idea that depleting V1 IN inhibition causes a preferential degradation of inhibition to TA flexor motor neurons. The increase in TA burst duration also caused a moderate lengthening of the step cycle period in V1 IN-ablated mice (Figure 5D). V2b IN-depleted mice displayed a strikingly different pattern of EMG activity during airstepping. In addition to an aggregate slowing of the motor rhythm, the duration of GS extensor muscle activity was markedly elongated (Figure 5C–E) when compared to control and V1 IN-ablated animals. This slowing of the motor rhythm following the loss of V2b INs can largely be attributed to the increased duration of GS extensor activity, as the TA flexor bursts were similar in length to those seen in control mice (Figure 5C, D). Taken together, these findings demonstrate that a flexor-dominant motor rhythm emerges when V1 INs are deleted, while the loss of V2b INs gives rise to an extensor-dominant motor rhythm. Further analysis of the EMG profiles of V1- and V2b-IN-ablated mice revealed a significant increase in the number and frequency of skipped or deleted GS and TA bursts in air-stepping juvenile mice (Figure 5A, arrowheads). Moreover, quantification of these deletions revealed a strong bias in their valency (Figure 5D). Whereas TA deletions were prevalent in V2b IN-ablated pups (Figure 5A, right panel, arrowheads), mice lacking V1 INs displayed a strong bias toward deletions in GS EMG activity (Figure 5, middle panel, arrowhead). The increased frequency of deletions in the V1- and V2b-IN-ablated mice (Figure 5D) coupled with the changes in phase duration (Figure 5C), strongly suggest that inhibition from V1 and V2b INs facilitates the transition from swing to stance and from stance to swing, respectively. Our observation that the deletions in both V1- and V2b-IN-ablated mice fall into the non-resetting category strongly suggests that both of these genetically defined cell populations are involved in pattern formation rather than rhythm generation (Lafreniere-Roula and McCrea, 2005). In order to examine the nature of any persistent changes to locomotor behavior that arise from the loss of V1 and V2b INs, we used combined kinematics and EMG recordings to quantitatively analyze changes in the gait of adult animals 3–5 weeks post DTX treatment. By this time, V1- and V2b-IN-ablated mice had regained partial control of their hindlimbs (see ‘Discussion’) and were able to use them to bear weight. V1 IN-ablated mice still displayed marked deficits in their gait, including a persistent hyperflexion of the hindlimbs during walking, which was particularly prominent in the mid-swing and early stance phases of the step cycle (Figure 6A, B, middle panel). Furthermore, V1 IN-ablated mice flexed their hindlimbs faster during the swing phase indicating that they are no longer able to properly modulate the speed and/or force of their flexion movements (Figure 6C). The EMG analysis of V1 IN-ablated mice revealed marked changes in the duration and co-activity of TA and GS muscle activity. These included (1) a persistent broadening of TA muscle activity, (2) an overlap in GS and TA burst activity during the transition from stance to swing, and (3) an increase in GS activity at the end of the stance phase (Figure 6D, middle panel, asterisk; Figure 6—figure supplements 1,2). The increase in GS EMG activity during stance phase appears to be a late behavioral modification that facilitates the opening of the ankle joint during walking, as it is not seen during the early phase of V1 IN ablation (see Figure 6—figure supplement 2). The co-activation of the TA and GS muscles was even more pronounced during swimming with EMG analysis revealing synchronous TA and GS muscle activity (Figure 6E, middle panel, asterisk) that causes the extension of the hindlimbs to be aborted during the power stroke. One factor that may contribute to the synchronous flexor–extensor activity seen during swimming may be the reduction in sensory feedback from GTOs that has been shown to occur (Akay et al., 2014). In V1 IN-ablated mice that lack sensory feedback gated by the V1 INs, the likely loss of Ib-mediated sensory inhibition during swimming could account for the observed co-activation of flexor–extensor muscles and abortive hindlimb movements (Figure 6, Figure 6—figure supplement 2; see ‘Discussion’). In addition to the hyperflexion phenotype, V1 IN-ablated mice displayed a prominent rear paw overshoot during walking (Figure 6—figure supplement 3). Kinematic analysis of walking V1 IN-ablated mice revealed two factors that contribute to rear paw overshoot: (1) an increase in the speed and degree of ankle flexion movements during swing (Figure 6C) and (2) an increase in the angular rotation of the pelvis during stepping (Figure 6—figure supplement 3). This rotation of the pelvis appears to be a behavioral adaption that helps extend the forward throw of the leg during late swing phase, so as to negate the reduced hindlimb extension that occurs when V1 INs are depleted from the spinal cord. In contrast to V1 IN-ablated mice, the locomotor phenotype that persists in mice lacking V2b INs was much milder, with the most notable change being a prolongation of the stance phase that results in overextension of the ankle joint during walking (Figure 6A, right panel, arrowhead). EMG analysis revealed the likely cause of this delay, namely a second ectopic burst of GS motor activity as the limb is transitioning from stance to swing (Figure 6D, right panel, asterisk). The delay in initiating flexion was not observed when the mice were swimming nor did we detect any marked differences in TA and GS EMG activity between control and V2b IN-ablated mice during swimming (Figure 6E, right panel). A further set of experiments were performed to probe the nature of the early motor deficits that arise when V1 INs are ablated in adult mice. Unfortunately, we were unable to perform a similar analysis of the early motor deficits that occur in adult V2b IN-ablated animals, as treating these animals with a single high dose of DTX caused bowel blockage and increased morbidity. This is likely to be due to the expression of Gata3 in a subset of enteric neurons. Whereas control mice (n = 5 animals) displayed a highly reproducible pattern of EMG activity both before and after DTX treatment, with the iliopsoas (IP) and TA muscles only being active during the swing phase of the step cycle (Figure 6—figure supplement 2, upper left panel), the depletion of V1 INs (n = 6 animals) resulted in ectopic IP and TA muscle EMG activity within 5 days of DTX treatment (Figure 6—figure supplement 2B, upper panel). This ectopic TA and IP muscle activity was even more pronounced 7 days after DTX treatment (Figure 6—figure supplement 2C, upper panel), with the TA muscle being co-active during stance with the GS muscle (see bars). A similar expansion of IP activity was observed, although to a less extent than that seen in the TA (Figure 6—figure supplement 2C, arrowhead). We posit that the co-activation of the TA and IP muscles during stance constitutes the underlying mechanism that restricts the hip and ankle joints from opening during walking, and it also contributes to the postural changes that occur in V1 IN-ablated mice following DTX treatment. By contrast, there was no increase in GS activity during the swing/flexor phase indicating the loss of V1 INs has an effect on ankle flexor but not extensor activity during walking. A careful comparison of kinematic movements (Figure 6—figure supplement 2, lower panels) and the time locked EMG traces (Figure 6—figure supplement 2, upper panels) revealed a close correspondence between angular movement of the ankle and EMG activity in the TA and GS muscles. During the early phase of V1 IN cell loss (5 days p. i.), the ankle joint angle at maximal flexion was reduced from 40° to less than 30° (lower middle panel). There was also a progressive decrease in maximal extension of the ankle joint in early swing phase (Figure 6—figure supplement 2, lower panels, asterisks). A similar hyperflexion phenotype was noted for the knee and hip joints, as indicated by their reduced angular changes during walking (Figure 6—figure supplement 2, lower panels). In the case of the hip joint, this is consistent with the observed expansion of IP muscle activity (Figure 6—figure supplement 2C, upper panel, arrowhead). In summary, our findings reveal that V1 INs function to (1) restrict flexor motor activity and promote active limb extension during stance and (2) moderate flexion movements during the swing phase of the step cycle. The weakening of ankle extensor inhibition in V2b IN-ablated mice, as indicated by ectopic GS burst activity during the swing phase of the step cycle and hyperextension of the ankle during walking (Figure 6), led us to ask whether V2b INs preferentially inhibit extensor–motor activity. To test this, we took advantage of the neonatal spinal cord preparation, which can be induced to produce fictive locomotion in vitro (Figure 7A, Kiehn, 2006; Goulding, 2009) and is amenable to optogenetic manipulation (Hagglund et al., 2010). Spinal cords from P0-P1 Gata3Cre; R26lsl-ChR2 (Ai32) pups displayed a characteristic alternating pattern of L2 flexor-related and L5 extensor-related locomotor rhythm in the presence of NMDA and 5-HT (Figure 7B–D, n = 6/6 cords). However, upon activating channelrhodopsin in V2b INs, there was a strong and highly selective decrease in extensor-related motor activity as measured by extracellular recordings from the L5 ventral root (Figure 7B–D, bar). By contrast, L2 flexor-related motor activity remained largely unchanged following V2b IN activation, although there was some disturbance in the coherence of the motor rhythm. Activation of V1 INs had a more pronounced effect on the motor rhythm, in that it completely suppressed L2 flexor-related and L5 extensor-related rhythmic bursting (Figure 7E, bar, n = 5/5 cords). This is likely to be due to the activation of Renshaw cells, which are known to strongly inhibit motor neurons (Windhorst, 1996; Bhumbra et al., 2014). In summary, the suppression of extensor-related L5 motor activity in the in vitro spinal cord preparation when V2b INs are activated provides strong evidence that V2b INs can functionally inhibit extensor-related motor activity during locomotion. This finding is consistent with the expansion of extensor motor neuron activity that we see following V2b IN ablation (Figures 5,6). 10. 7554/eLife. 04718. 015Figure 7. Effect of optogenetic activation of V2b and V1 INs on in vitro locomotion. (A) Schematic showing the in vitro recording setup used for the localized light activation of ChR2 in V1 and V2b INs. (B–D) Representative ENG recordings from a P0 Gata3Cre; R26lsl-ChR2 (Ai32) spinal cord showing the suppression of L5 extensor-related activity in the presence of blue light photostimulation (blue bar). The lumbar levels that were photostimulated are indicated as upper (L1–L2), mid (L3–L4), and lower (L5–L6). (E) Representative recording from a P0 En1Cre; R26lsl-ChR2 (Ai32) spinal cord showing suppression of L2 and L5 ventral root activity following photostimulation of V1 INs at L3–L4. Upper traces represent the raw filtered ENG recordings. Lower traces display the matching online rectified ENG signal. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 01510. 7554/eLife. 04718. 013Figure 8. Biased V1 and V2b connections onto flexor- and extensor-related motor neurons. (A) Schematic of experimental design used for labeling V1 and V2b inhibitory contacts on specific motor pools. Motor neurons (MNs) were retrogradely labeled by injecting Cy5-CTB into individual hindlimb muscles at P11. Interneuron class-specific contacts were labeled with a conditional Thy1lsl-YFP reporter allele (green) and inhibitory contacts were detected with antibodies to vGAT and GlyT2 (red). (B) Putative V1- and V2b-derived inhibitory contacts (vGAT/GlyT2 (red) and Thy1-YFP reporter (green) ) onto CTB-labeled TA and GS motor neurons (blue). Filled arrowheads indicate examples of inhibitory synaptic terminals that co-localize with YFP. YFP-negative inhibitory contacts are marked with open arrowheads. (C) Quantification of V1- or V2b-IN-derived inhibitory contacts on the soma/proximal dendrites of defined hindlimb motor neurons (n = 12 motor neurons per pool). A greater number of putative V1 IN inhibitory synapses contact flexor motor neuron pools as compared to their antagonist extensor-related motor pools. In contrast, V2b-derived contacts represent a greater proportion of the inhibitory synapses onto extensor-related motor neurons as compared to antagonist flexor-related motor neurons. Abbreviations: GS, gastrocnemius; TA, tibialis anterior. Error bars: mean ± s. d. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 01310. 7554/eLife. 04718. 014Figure 8—figure supplement 1. Axonal projections of V1 and V2b INs. P0 En1Cre; R26lsl-HTB and Gata3Cre; R26lsl-HTB mice were used to label and trace the projections of V1 and V2b INs, respectively. Rhodamine-conjugated dextran was applied unilaterally to the cut ventral horn at the L2. Data represent cumulative cell counts from two cords each for V1 and V2b INs. The distribution shown covers the lumbar spinal cord from the T13/L1 border to L6. DOI: http: //dx. doi. org/10. 7554/eLife. 04718. 014 In view of the opposing phenotypes that arise from ablating V1 and V2b INs, we asked if differences in the innervation of hindlimb extensor and flexor motor pools by these two classes of inhibitory neuron might contribute to their opposing actions on limb flexion and extension movements. To address this question, defined hindlimb motor pools were visualized by backfilling them from their respective muscles with Cholera Toxin-B (CTB). A conditional Thy1lsl-YFP transgene reporter (Buffelli et al., 2003) was then used in combination with En1Cre or Gata3Cre to label the terminal processes of V1 and V2b INs. Presumptive inhibitory synapses on CTB-labeled motor neurons were then identified with antibodies to vGAT and GlyT2 (Figure 8A). The analysis of V1 and V2b inhibitory contacts focused on the soma and proximal dendrites of motor neurons, which is where inputs from inhibitory interneurons such IaINs and Renshaw cells tend to be concentrated (Jankowska and Roberts, 1972; Brown, 1981). Counts of V1-derived inhibitory contacts on motor neurons revealed a strong positive bias in their innervation of the TA ankle flexor motor neurons (Figure 8). This preferential innervation is also seen for hip flexor motor neurons, which receive a greater percentage of inputs from V1 INs when compared to their antagonist extensor motor pools (Zhang et al., 2014). In comparing V1 inhibitory inputs onto TA and GS motor neurons, we counted an approximate twofold greater number of V1-derived synaptic contacts onto TA motor neurons as compared to their antagonist GS counterparts (Figure 8B, C). Conversely, the proportion of inhibitory contacts onto GS (ankle) extensor motor neurons from V2b INs was higher than the proportion of V2b contacts onto their TA counterparts (Figure 8C). We have not seen any significant difference in the proportion of V2b IN contacts onto the knee quadriceps (Q) and BF/St motor pools (Zhang et al., 2014). These two knee muscle groups are bifunctional (Yakovenko et al., 2002), and the near neutral weighting of V2b IN inputs onto these motor pools may reflect their bi-functional nature with regard to knee and hip flexion-extension movements. In summary, our findings reveal an overall asymmetry in V1 and V2b in inputs, with flexor-related motor pools receiving proportionately fewer inputs from V2b INs and more from V1 INs, while V2b INs exhibit a bias toward extensor-related motor pools. Multiple methods have been implemented to inactivate genetically defined neurons within a circuit (Tan et al., 2006; Luo et al., 2008; Kim et al., 2009). These include blocking neurotransmission with tetanus toxin (Yu et al., 2004; Zhang et al., 2008,2014) and suppressing neuronal excitability with heterologous chloride channels or G-protein-coupled receptors (GPCRs) that activate GIRK channels (Gosgnach et al., 2006; Tan et al., 2006; Armbruster et al., 2007; Ray et al., 2011). These systems all have drawbacks. Approaches using tetanus toxin are often non-inducible, while ligand-mediated silencing using Gi-coupled receptors can be highly variable and difficult to quantify. By contrast, DTR-mediated neuronal ablation has the advantage of being highly quantifiable, and the timing of cell killing can also be controlled to minimize potential developmental changes due to the chronic silencing or ablation of cells at early developmental times (Gosgnach et al., 2006; Crone et al., 2008; Zhang et al., 2008). Most importantly, by using an intersectional approach to restrict DTR expression to the caudal CNS, we were able to spare essential functions such as respiration and chewing. With further refinements, this intersectional system can be used to target and ablate discrete populations of neurons, including genetically defined subsets of V1 and V2b INs. Our results demonstrate that V1 INs facilitate the transition from swing to stance, and V2b INs facilitate the transition from stance to swing. In the Xenopus tadpole, V1 (aIN) cells promote the transition between the active and inactive phases of the swimming rhythm by providing early phase inhibition to motor neurons (Li et al., 2004). The CiA cells, which are homologous to the V1 INs, are likely to function in a similar fashion in zebrafish (Higashijima et al., 2004). Interestingly, we see an increase in the incidence of GS (extensor) EMG deletions in airstepping mice that lack V1 INs, suggesting the transition from flexion to extension is compromised (Figure 5). Interestingly, these deletions, and the flexor deletions that arise from the loss of the V2b INs, resemble non-resetting deletions in the cat and turtle (Grillner and Zangger, 1979; Lafreniere-Roula and McCrea, 2005; McCrea and Rybak, 2008; Stein, 2008). The absence of resetting deletions suggests both populations belong to the pattern forming, rather than rhythm generating, layer of the locomotor CPG. Moreover, the opposing nature of the deletions that occur when V1 vs V2b INs are inactivated suggests that the differential silencing of these two inhibitory cell populations may contribute to the valence of corrective reflexes such as the stumbling corrective reaction (Forssberg, 1979) and crossed extension reflex (Sherrington, 1910). Our preliminary anatomical analysis of the organization of V1 and V2b inputs to motor neurons (Figure 8) reveals a genetically encoded bias in the innervation of flexor vs extensor motor pools by V1 and V2b INs (see also Zhang et al., 2014). Although we were unable to score inhibitory contacts on the distal dendrites of motor neurons, studies showing IaIN and Renshaw cell inhibitory synapses are preferentially located on the soma, and proximal dendrites of motor neurons (Brown, 1981; Fyffe, 1991) suggests that the counts we obtained in this study are likely to be a reasonable measure of the density of V1 and V2b inhibitory synaptic contacts on motor neurons. This in turn suggests that relative differences in the inhibitory drive to motor neurons from the V1 and V2b IN populations may contribute to the opposing actions of V1 and V2b INs on flexor–extensor movements. Our finding that ChR2 activation of V2b INs in the isolated spinal cord selectively suppresses L5 extensor-related motor neuron activity (Figure 7) is consistent with such a model. In considering the differential innervation of motor neurons by V1 and V2b INs, we would like to suggest that there are multiple advantages in having a system where biased rather than segregated inhibitory inputs from V1 and V2b INs determine the valence of flexor–extensor movements. First, biased inputs from the V1 and V2b INs would facilitate the graded activation of synergist and antagonist motor neurons. This graded recruitment of antagonist flexor–extensor motor neurons would be expected to play an important role in modulating limb stiffness and compliance, which is necessary for smooth movements and postural control. Second, the co-innervation of flexor and extensor motor neurons by V1 and V2b INs would enable motor neurons to actively summate and compare inhibitory inputs that are differentially gated by these two inhibitory interneuron populations. There is growing evidence that motor neurons receive a mixture of tonic and dynamic inhibition during locomotion that together with excitatory inputs regulate motor neuron excitability (Berg et al., 2007; Johnson et al., 2012). In particular, the altered membrane conductances produced by concurrent excitation and inhibition are believed to be a fundamental mechanism for changing the gain and dynamic properties of neurons (Chance et al., 2002; Mitchell and Silver, 2003; Abbott and Chance, 2005). Consequently, gain modulation and increases in the variability of motor neuron spiking represent an important mechanism for altering the dynamic range of motor neuron activity, so as to produce smooth gradients of muscle force transduction (Kristan, 2007; Johnson et al., 2012). Johnson et al. (2012) have recently shown that inhibitory IaINs are a major source of the tonic inhibitory drive underlying the push–pull control of motor activity in ankle extensor motor neurons. This tonic inhibition, in concert with tonic excitation, causes a net increase in the force modulation produced by ankle extensor muscles. Our observation that V1 and V2b INs are the sole source of Ia inhibition to motor neurons (Zhang et al., 2014) suggests that they make a major contribution to inhibitory push–pull conductances. As such, the biased innervation of motor neurons by V1 and V2b INs (Figure 8) could facilitate push–pull in limb motor neurons under a range of behavioral conditions. For example, in situations where V2b INs are being dynamically activated by cutaneous feedback, the V1 INs, or subsets thereof, might be tasked with providing tonic inhibition to motor neurons. Our in vivo behavioral analyses showing an inhibitory network comprised of either V1 or V2b INs still generates an alternating pattern of flexor–extensor activity in awake behaving mice, albeit an abnormal one, concurs with our in vitro analysis showing flexor–extensor alternation is only completely degraded when both the V1 and V2b IN populations are inactivated (Zhang et al., 2014). These findings argue that the V1 and V2b INs can function in a redundant manner to produce a grossly alternating flexor–extensor locomotor output, and they are in general agreement with the mixed innervation of flexor and extensor motor neurons by V1 and V2b INs (Figure 8; Zhang et al., 2014). The co-innervation of flexor and extensor motor neurons by V1 and V2b INs may contribute to the functional recovery that occurs following V1 or V2b IN ablation. This functional recovery is suggestive of a degree of plasticity in the inhibitory control of flexor–extensor movements by both inhibitory populations. Interestingly, the functional deficits that persist after ablating the V1 INs are more pronounced than those found following V2b IN ablation. This might be attributable to the relative abundance of these two cell types in the lumbar spinal cord, where there are twice as many V1 INs as compared to V2b INs (Zhang et al., 2014). Differences in the axonal morphology of V1 and V2b INs may also contribute to the persistent hindlimb hyperflexion phenotype V1 IN-ablated mice display, as the motor pools innervating hip and ankle flexors tend to be skewed rostrally in the lumbar spinal cord when compared to their extensor antagonist motor pools (McHanwell and Biscoe, 1981; Yakovenko et al., 2002). In this context, it is worth noting that V2b INs predominantly project their axons caudally (Figure 8—figure supplement 1) and may therefore have a limited capacity to substitute for those V1 INs that have rostrally projecting axons. In addition to this, there are fewer inhibitory contacts from V2b INs onto flexor motor neurons (Figure 8; Zhang et al., 2014). By contrast, the V2b IN-ablated mice regain a large measure of normal limb movement in the aftermath of V2b IN ablation. In this instance, V1 INs, many of which project caudally (Figure 8—figure supplement 1), may compensate for the initial loss of V2b IN-derived inhibitory inputs that are proportionately more abundant on extensor motor neurons. The one locomotor deficit that persists when the V2b INs are removed is the delay in the transition from stance to swing during walking (Figure 6). This transition is controlled in part by Ib pathways from ankle extensor GTOs (Duysens and Pearson, 1980; Pearson, 2008). Our characterization of presynaptic inputs to V1 and V2b INs showing the V2b INs receive inputs from neurons in the dorsal horn (F Stam and MG, unpublished findings) is a strong indication that inhibitory IbINs are derived from the V2b IN population. As such, the presumed loss of inhibitory IbINs that occurs when V2b INs are ablated could account for the delayed transition from stance to swing that we observe (Pearson, 2008). The loss of IbINs in the V2b IN-ablated mice might also explain why these mice do not show any major change in GS and TA muscle activity during swimming, as Golgi tendon-derived sensory feedback is normally attenuated during this activity (Akay et al., 2014). In summary, this study demonstrates that V1 and V2b INs differentially control flexor–extensor motor output, with V1 INs suppressing flexor motor activity during the stance/extension phase of walking to ensure proper extension of the limbs, while the V2b INs suppress extensor activity to facilitate limb extension and ensure the timely transition from stance to swing. Our results are consistent with the V1 and V2b INs contributing to the dynamic control of limb movements during walking via their differential effects on flexor and extensor motor activity. It should be noted that the V1 and V2b IN populations are made up of multiple cell types and the respective contribution that each of these cell types make to flexor–extensor-driven behaviors needs to be assessed. Future efforts to evaluate in depth how these two populations control flexor–extensor motor behaviors will require a better understanding of the molecular, anatomical, and physiological diversity that exists within these two inhibitory IN populations, coupled with a detailed functional characterization of V1 and V2b IN subtypes. | Title: A genetically defined asymmetry underlies the inhibitory control of flexor-extensor locomotor movements Summary: Although there are many different movements an animal can make with its limbs-from reaching to walking-they all basically involve two sets of muscles that act as opposing levers around each joint. 'Flexor' muscles contract to bend the limb, and 'extensor' muscles contract to extend the limb. When an animal is walking these two sets of muscles contract repeatedly, one after the other. Inhibitory neurons in the spinal cord coordinate these walking movements by preventing the flexor or extensor muscles from contracting at the same time. In 2014, researchers discovered that two groups of inhibitory neurons, known as the V1 and V2b interneurons, are essential for this alternating pattern of flexing and extending of the limbs of newborn mice. However, these experiments were not able to assess the particular contribution that the V1 and V2b neurons each make to limb movements. Now, Britz et al. -including several of the researchers involved in the 2014 study-have used a sophisticated genetic technique in mice to investigate the role that each group of neurons plays separately. This involved introducing a gene into either the V1 or V2b neurons that makes them susceptible to being killed with the diphtheria toxin. Injecting the mice with diphtheria toxin selectively removed these cells from the regions of the spinal cord that controls hindlimb movements. Britz et al. found that removing either group of neurons prevented the mice from walking normally. Eliminating the V1 neurons caused extreme flexing of the hindlimbs, revealing that the V1 neurons are needed to extend the limb by inhibiting the motor neurons that contract the flexor muscles. In contrast, the loss of V2b neurons caused exaggerated hindlimb extension, indicating that the V2b neurons inhibit the motor neurons that innervate extensor muscles. Both the V1 and V2b groups of neurons contain a wide range of different cell types. Future studies will therefore need to explore how these different cells are involved in coordinating the motions involved in walking. | 13,980 | 478 | lay_elife | en |
Write a title and summarize: La légalité a-t-elle été dépassée au nom de l’ordre républicain? La question a été posée à huis clos, jeudi 13 septembre, dans la chambre de l’instruction de Lyon à propos d’une journée particulière sur l’emblématique place Bellecour. Rappel des faits : le 21 octobre 2010, des manifestants s’apprêtent à défiler pour protester contre l’allongement de la durée de cotisation pour les retraites. Les autorités décident d’encercler hermétiquement les lieux. Pendant plus de six heures, quelque 600 personnes sont totalement bloquées et s’ensuivent des dizaines de contrôles d’identité. Choqués par ce traitement policier, dix-neuf associations et syndicats ont porté plainte dans la foulée de la manifestation. Les témoins ont eu l’impression de vivre « une garde à vue à ciel ouvert ». Après classement du parquet, ils ont porté plainte avec constitution de partie civile devant le doyen des juges d’instruction, qui a prononcé un non-lieu. Ils ont alors fait appel, demandant à la chambre de l’instruction d’engager des poursuites et de mettre en examen Jacques Gérault et Albert Doutre, à l’époque préfet et directeur départemental de la sécurité publique. « Détention arbitraire » « Cette opération est parfaitement illégale, sans aucune réquisition, sans fondement. Elle relève de la détention arbitraire, la police ne peut pas s’arroger tous les droits dans une société démocratique », estime l’avocat Bertrand Sayn. Il rappelle que le Défenseur des droits, dans un rapport de 2017, a eu l’occasion de dénoncer « le cadre légal très incertain, voire inexistant » des techniques de maintien de l’ordre consistant à retenir des groupes dans une nasse. Citant en exemple La Manif pour tous, le Défenseur des droits a déploré des durées prolongées de rétention collective « à l’encontre de manifestants pacifiques ». « Cette opération était exceptionnelle mais elle répondait à une situation complexe de grand péril ; elle est parfaitement légitime et légale », soutient au contraire Gabriel Versini, le défenseur de l’ancien directeur de la police lyonnaise. « La ville était la proie de violences sans précédent, faites d’émeutes, de pillages, d’affrontements incessants avec la police, selon des techniques de guérillas urbaines. Des groupes ont été repérés quelques heures avant la manifestation. » Arrêt fébrilement attendu L’avocat cite une décision de la Cour européenne des droits de l’homme (CEDH) du 15 mars 2012 reconnaissant la technique policière du kettling (littéralement « enchaudronnement ») consistant à cerner et à bloquer des attroupements pour éviter des violences imminentes. Pour la CEDH, la police britannique avait légitimement assuré la sécurité lors de la manifestation altermondialiste d’Oxford Circus, le 1er mai 2001, marquée par l’irruption de groupes très violents. Néanmoins, l’arrêt Austin, du nom de la citoyenne britannique concernée, dit aussi que le kettling ne doit pas servir de prétexte pour « indirectement étouffer ou décourager des mouvements de protestation ». | Title: A Lyon, la justice examine la légalité d'une opération d'encerclement Summary: En 2010, lors d'une manifestation, plus de 600 personnes avaient été encerclées par la police pendant plusieurs heures sur la place Bellecour. Des associations et syndicats se battent depuis pour que des poursuites soient engagées à l'encontre du préfet et du directeur départemental de la sécurité publique de l'époque. | 737 | 97 | mlsum_fr | fr |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Community Recovery and Enhancement Act of 2011'' or the ``CRE Act of 2011''. SEC. 2. DEDUCTION FOR CERTAIN PAYMENTS MADE REDUCE DEBT ON COMMERCIAL REAL PROPERTY. (a) In General.--Part VI of subchapter B of chapter 1 of the Internal Revenue Code of 1986 (relating to additional itemized deductions for individuals and corporations) is amended by adding at the end the following new section: ``SEC. 199A. DEDUCTION FOR PAYMENTS MADE TO REDUCE DEBT ON COMMERCIAL REAL PROPERTY. ``(a) In General.--There shall be allowed as a deduction an amount equal to 50 percent of any qualified debt reduction payment made during the taxable year by the taxpayer with respect to qualified indebtedness on eligible commercial real property held by the taxpayer. ``(b) Maximum Deduction.--The deduction allowed by subsection (a) for any taxable year shall not exceed, with respect to each eligible commercial real property, whichever of the following amounts is the least: ``(1) The amount equal to 50 percent of the excess (if any) of-- ``(A) the amount of the qualified indebtedness secured by such property as of the beginning of such taxable year (reduced by amounts required to be paid under the terms of the loan during such taxable year), over ``(B) 50 percent of the fair market value of such property as of the close of the taxable year. ``(2) $10,000,000. ``(3) The adjusted basis of such property as of the close of such taxable year (determined without regard to qualified debt reduction payments made during the taxable year and depreciation for such year). ``(c) Eligible Commercial Real Property.--For purposes of this section, the term `eligible commercial real property' means any commercial real property if-- ``(1) as of the beginning of the taxable year, the amount of the qualified indebtedness secured by such property is at least equal to 85 percent of the fair market value of the property, or ``(2) such property is, or is reasonably expected to be, treated as being in an in-substance foreclosure by the Comptroller of the Currency. ``(d) Qualified Debt Reduction Payment.--For purposes of this section, the term `qualified debt reduction payment' means the amount of cash paid by the taxpayer during the taxable year to reduce the principal amount of qualified indebtedness of the taxpayer but only to the extent such amount exceeds the amounts required to be paid under the terms of the loan during such taxable year. ``(e) Property Held by a Partnership.-- ``(1) In general.--In the case of property held by a partnership, a qualified debt reduction payment by the partnership may be taken into account under this section only if-- ``(A) such payment is attributable to a qualified equity investment made by a partner in such partnership, and ``(B) any deduction under this section which is attributable to such investment is properly allocated to such partner under section 704(b). ``(2) Qualified equity investment.--For purposes of this section-- ``(A) In general.--The term `qualified equity investment' means any equity investment (as defined in section 45D(b)(6)) in a partnership if-- ``(i) such investment is acquired by the partner at its original issue (directly or through an underwriter) solely in exchange for cash, ``(ii) at least 80 percent of such cash is used by the partnership to reduce the principal amount of qualified indebtedness of the partnership, ``(iii) the portion of such cash not so used is used by the partnership for improvements to commercial real property held by the partnership, and ``(iv) the person or persons otherwise entitled to depreciation with respect to the portion of the basis of the property being reduced under subsection (g)(1) consent to such reduction. ``(B) Redemptions.--A rule similar to the rule of section 1202(c)(3) shall apply for purposes of this paragraph. ``(f) Other Definitions.--For purposes of this section-- ``(1) Qualified indebtedness.--The term `qualified indebtedness' means any indebtedness-- ``(A) which is incurred or assumed by the taxpayer on or before January 1, 2009, and ``(B) which is secured by commercial real property held by the taxpayer at the time the qualified debt reduction equity payment is made by the taxpayer. ``(2) Commercial real property.--The term `commercial real property' means section 1250 property (as defined in section 1250(c)); except that such term shall not include residential rental property (as defined in section 168(e)(2)) unless the building contains at least 3 dwelling units. ``(g) Application of Section 1250.--For purposes of determining the depreciation adjustments under section 1250 with respect to any property-- ``(1) the deduction allowed by this section shall be treated as a deduction for depreciation, and ``(2) the depreciation adjustments in respect of such property shall include all deductions allowed by this section to all taxpayers by reason of reducing the debt secured by such property. ``(h) Special Rules.-- ``(1) Basis reduction.--The basis of any property with respect to which any qualified debt reduction payment is made shall be reduced by the amount of the deduction allowed by this section by reason of such payment. ``(2) Refinancings.--The indebtedness described in subsection (f)(1)(A) shall include indebtedness resulting from the refinancing of indebtedness described in such subsection (or this sentence), but only to the extent it does not exceed the amount of the indebtedness being refinanced. ``(3) Denial of deduction for debt-financed payments.--No deduction shall be allowed by this section for any qualified debt reduction payment-- ``(A) to the extent indebtedness is incurred or continued by the taxpayer to make such payment, and ``(B) in the case of a qualified debt reduction payment made by a partnership on qualified indebtedness on commercial real property held by the partnership, to the extent of indebtedness-- ``(i) which is incurred or continued by any partner to whom such payment is allocable, and ``(ii) which is secured by such partner's interest in the partnership or by such commercial real property. ``(4) Treatment of amounts required to be paid by reason of loan default.--For purposes of subsections (b)(1)(A) and (d), accelerated payments required to be made under the terms of a loan solely by reason of a default on the loan shall not be taken into account. ``(5) Recapture of deduction if additional debt within 3 years.-- ``(A) In general.--If a taxpayer incurs any additional debt within 3 years after the date that the taxpayer made a qualified debt reduction payment, the ordinary income of the taxpayer making such payment shall be increased by the applicable percentage of the recaptured deduction. ``(B) Recaptured deduction.--For purposed of this paragraph, the recaptured deduction is the excess of-- ``(i) the deduction allowed by subsection (a) on account of a qualified debt reduction payment, over ``(ii) the deduction which would have been so allowed if such payment had been reduced by the additional debt. ``(C) Applicable percentage.--The applicable percentage shall be determined in accordance with the following table: ``If, of the 3 years referred to in The applicable subparagraph (A), the additional percentage is: debt occurs during the: 1st such year...................................... 100 2d such year....................................... 66 2/3 3d such year....................................... 33 1/3. ``(D) Partnerships.-- ``(i) Allocation of income inclusion.--Any increase in the ordinary income of a partnership by reason of this paragraph shall be allocated (under regulations prescribed by the Secretary) among the partners receiving a deduction under this section by reason of making qualified equity investments in the partnership. ``(ii) Debt-financed equity investment by partner.--Rules similar to the rules of the paragraph shall apply in cases where additional debt is incurred by a partner making a qualified equity investment in a partnership. ``(E) Subsequent deprecation.--The deductions under section 168 for periods after a recaptured deduction under this paragraph shall be determined as if the portion of the qualified debt reduction payment allocable to the recaptured deduction had never been made. ``(6) Recapture where partner disposes of interest in partnership.--If any partner to whom a deduction under this section is allocable by reason of making a qualified equity investment in a partnership disposes of any portion of such partner's interest in the partnership within 1 year after the date such investment was made, the ordinary income of such partner shall be increased by the amount which bears the same ratio to the deduction allowed on account of such investment as such portion bears to such partner's interest in the partnership immediately before such disposition. ``(7) Exemption from passive loss rules.--Section 469 shall not apply to the deduction allowed by this section. ``(i) Application of Section.--This section shall apply to qualified debt reduction payments made within the 2-year period beginning on the day after the date of the enactment of this section.''. (b) Earnings and Profits.--Subsection (k) of section 312 of such Code is amended by adding at the end the following new paragraph: ``(6) Treatment of section 199a.--Paragraphs (1) and (3) shall not apply to the deduction allowed by section 199A.''. (c) Clerical Amendment.--The table of sections for part VI of subchapter B of chapter 1 of such Code is amended by adding at the end the following new item: ``Sec. 199A. Deduction for payments made to reduce debt on commercial real property.''. (d) Effective Date.--The amendments made by this section shall apply to taxable years ending after the date of the enactment of this Act. | Title: To amend the Internal Revenue Code of 1986 to allow a deduction for certain payments made to reduce debt on commercial real property Summary: Community Recovery and Enhancement Act of 2011 or the CRE Act of 2011 - Amends the Internal Revenue Code to allow a tax deduction for payments made to reduce debt on eligible commercial property. Limits the amount of such deduction to the lesser of: (1) 50% of the excess of the amount of qualified debt secured by such property, (2) 50% of the fair market value of such property, (3) $10 million, or (4) the adjusted basis of such property at the close of the taxable year. Defines "eligible commercial property" as any commercial real property if: (1) the amount of the qualified indebtedness secured by such property is at least 85% of the fair market value of the property, or (2) such property is, or is reasonably expected to be, treated as being in an in-substance foreclosure by the Comptroller of the Currency. Denies a tax deduction for debt reduction payments that are debt-financed. Requires a recapture in income of tax deduction amounts allowed by this Act if additional indebtedness is incurred within three years after a qualified debt reduction payment is made. | 2,457 | 293 | billsum | en |
Write a title and summarize: Type I collagen-containing fibrils are major structural components of the extracellular matrix of vertebrate tissues, especially tendon, but how they are formed is not fully understood. MMP14 is a potent pericellular collagenase that can cleave type I collagen in vitro. In this study, we show that tendon development is arrested in Scleraxis-Cre: : Mmp14 lox/lox mice that are unable to release collagen fibrils from plasma membrane fibripositors. In contrast to its role in collagen turnover in adult tissue, MMP14 promotes embryonic tissue formation by releasing collagen fibrils from the cell surface. Notably, the tendons grow to normal size and collagen fibril release from fibripositors occurs in Col-r/r mice that have a mutated collagen-I that is uncleavable by MMPs. Furthermore, fibronectin (not collagen-I) accumulates in the tendons of Mmp14-null mice. We propose a model for cell-regulated collagen fibril assembly during tendon development in which MMP14 cleaves a molecular bridge tethering collagen fibrils to the plasma membrane of fibripositors. Matrix metalloproteinase 14 (MMP14, also known as membrane type I-MMP) is a member of the family of MMPs and contains a transmembrane domain for insertion into the plasma membrane (Sato et al., 1994). MMP14 has been implicated in cancer cell invasion (Hotary et al., 2003) and embryonic development (Holmbeck et al., 1999; Zhou et al., 2000) because of its ability to degrade extracellular matrix (ECM) macromolecules especially type I collagen (Overall, 2001; Tam et al., 2002; Lee et al., 2006; Kessenbrock et al., 2010; Gialeli et al., 2011). Mice deficient in MMP14 die within a few weeks of birth with generalized connective tissue abnormalities including osteopenia and soft tissue frailty (Holmbeck et al., 1999; Zhou et al., 2000). We were curious why absence of MMP14, which is an efficient collagenase in vitro, leads to connective tissue frailty rather than collagen accumulation. Collagens are a large family of triple helical proteins that are widespread throughout the vertebrate body and are critical for tissue scaffolding (Huxley-Jones et al., 2007). More than 28 collagen and collagen-related proteins occur in vertebrate tissues of which type I collagen is the archetypal member of the subfamily of fibril-forming collagens (Kadler et al., 2007). The fibrils formed from type I collagen are the largest (with a mass per unit length up to ∼0. 3 TDa/µm) and most size pleomorphic (from ∼1 µm to >1 mm) protein polymers in vertebrates and are essential for fibrous tissue development (Schnieke et al., 1983). Collagen fibril assembly has best been studied in embryonic tendon, which contains narrow diameter (∼50 nm) fibrils in which one end of the fibril is located within actin-dependent (Canty et al., 2006) invaginations of the plasma membrane called fibripositors (Canty et al., 2004). Fibripositors are points of fibril assembly and sites of attachment of the cell to the ECM (Kalson et al., 2013) and exhibit a range of morphologies depending on the presence or absence of a slender finger-like projection of the plasma membrane. Protruding fibripositors exhibit the invagination and the finger-like projection whereas recessed fibripositors only exhibit the invagination (Kalson et al., 2013). The transport of collagen fibrils into fibripositors is powered by non-muscle myosin II and is not part of a fibril degradation process in embryonic tendon (Kalson et al., 2013). Short collagen fibrils can be found within membrane-sealed compartments termed fibricarriers (Canty et al., 2004; Kalson et al., 2013). The transition from a unimodal distribution of narrow (∼50 nm) diameter collagen fibrils in embryonic tendon to a bimodal distribution in adult tissues with means of ∼50 nm and ∼200 nm diameter fibrils is a fascinating phenomenon (Parry et al., 1978; Derwin and Soslowsky, 1999). The presence of narrow-diameter collagen fibrils in fibripositors during E14. 5 to birth (in mice) signifies stage 1 of tendon development during which fibril number is determined (Kalson et al., 2015). Stage 2 occurs soon after birth (in mice) and is characterized by the disappearance of fibripositors, the release of fibrils to the ECM and the growth of fibrils in length and diameter (Kalson et al., 2015). We show here that in the absence of MMP14, progression from stage 1–2 does not occur, fibrils are retained by fibripositors, fibril diameters keep to ∼50 nm, and tendon development stops at stage 1. Wild type (WT) and Mmp14 knockout (KO) littermates had similar birth weights but the KO mice were growth retarded at 3 days after birth (P3) (Figure 1A, as reported previously [Holmbeck et al., 1999]). Morphometric analyses at P0 showed that the tendons of the KO mice were thinner than those of WT mice (Figure 1B) and had fewer bundles of fibrils (Figure 1C, D). In transverse section, Mmp14 KO tendons had fewer fibrils that were organized into fewer and irregular bundles (Figure 1C, D). We detected no difference in the number of cells per unit volume of the tendon between WT and Mmp14 KO tendon (Figure 1—figure supplement 1A–C). There was no difference in fibril volume fraction (FVF) (Figure 1D); therefore, there was no evidence of abnormal fibril–fibril interactions. Mmp14 KO tendons were mechanically weaker than WT tendon (Figure 1E). However, after adjustment for size, the KO tendons had similar mechanical properties to those of tendons from WT animals, which was consistent with the normal FVF. Quantitative PCR analysis showed no differences in Col1a1 gene expression at P0 (Figure 1F) and [14C]-proline labeling showed that procollagen processing was unaffected by loss of MMP14 (at P7, Figure 1G). However, despite no differences in gene expression or procollagen processing with MMP14 deficiency we found decreased collagen synthesis in KO samples, compared to WT animals (Figure 1—figure supplement 1D). We next analyzed collagen fibril diameters. As shown in Figure 1H, the diameters in P0 KO mouse-tail tendon were significantly larger (by ∼12%) than those in WT mice. 10. 7554/eLife. 09345. 003Figure 1. Neonatal Mmp14-deficient mice have small and weak tendons. (A) Weight of wild type (WT) and Mmp14 knockout (Mmp14 KO) littermates at birth (P0) and at 3 days postnatal (P3). (B) The cross-sectional transverse area of P0 Mmp14 KO tendons is significantly smaller than WT tendons. (C) TEM images of P0 tail tendon demonstrate that KO tendons are smaller and show dysmorphic, enlarged bundles of collagen fibrils (arrowhead). Scale bars 5 μm. (D) KO tendons have fewer, larger fibril bundles, but the FVF is not different to WT tendons. (E) KO tendons are weaker than WT tendons but have normal mechanical properties after adjusting for differences in size. (F) Analysis of Col1a1 mRNA by qPCR in P0 tendons revealed no difference in gene expression. (G) 14C-proline labeling of collagen demonstrated normal collagen processing at P7 in WT and KO tail tendon. (H) Fibril diameter distributions of KO and WT tail tendon at P0 revealed significantly increased fibril diameters in KO mice. Bars show SEM. *p < 0. 05, †p > 0. 05 (t-tests). DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 00310. 7554/eLife. 09345. 004Figure 1—figure supplement 1. Cell number and type I collagen synthesis in neonatal WT and Mmp14 KO tail tendon. 3D reconstruction from SBF-SEM analysis of P0 (A) WT and (B) Mmp14 KO tendons. Green/turquoise, outline of the tendon. Red/pink spheres, cell nuclei. Scale bars 10 µm. (C) Estimated mean cell number per unit volume of tissue shows that the absence of MMP14 does not affect cell number at birth. Bars show SEM. †p > 0. 05 (t-test). (D) Densitometry data for P7 tail tendons were labeled with 14C-proline for 1 hr and separate extracellular and intracellular extracts prepared as described (Canty et al., 2004). A significant decrease in [14C]-collagen relative to β-actin (detected by western blotting) in the intracellular extracts was observed in the KO samples (*p < 0. 01, t-test). DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 004 We used transmission electron microscopy (TEM), serial block face-scanning electron microscopy (SBF-SEM), and serial section electron tomography to examine embryonic WT and Mmp14 KO tendons. We were careful to use tail tendon from anatomical site- and age-matched embryonic WT and KO mice. TEM analysis showed E15. 5 WT tendons contained collagen fibrils in the ECM and a small number of collagen fibrils in fibripositors (Figure 2A and Figure 2—figure supplement 1A). In contrast, Mmp14 KO tendons contained conspicuous electron-lucent invaginations characteristic of recessed fibripositors (Figure 2B and Figure 2—figure supplement 1B). There were ∼8 times the number of fibripositor cross-sections per nucleus in KO tenocytes (Figure 3—figure supplement 3C). Tracing of fibrils in 3D reconstructions from SBF-SEM and electron tomography (Figure 2—figure supplement 1C, D) showed the presence of deep recessed fibripositors in Mmp14 KO cells (Videos 1,2, respectively) with looped collagen fibrils. Figure 2—figure supplement 2 shows a diagrammatic representation of a typical recessed fibripositor containing looped collagen fibrils. 10. 7554/eLife. 09345. 005Figure 2. Fibricarrier analysis of wild-type, Mmp14 KO, and Col-r/r embryonic tail tendon. Tail tendons at E15. 5 of development from (A) wild-type, (B) Mmp14 KO, and (C) Col-r/r mice. Black arrowhead, recessed fibripositor (electron lucent) -containing collagen fibrils. White arrow, enclosed electron-dense compartment. Scale bars 500 nm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 00510. 7554/eLife. 09345. 006Figure 2—figure supplement 1. Mmp14-deficient mice have prominent recessed fibripositors. SBF-SEM of (A) WT and (B) Mmp14 KO tendons at E15. 5 showing fibripositors in WT tendons (blue box) and intracellular collagen fibril-containing compartments in Mmp14 KO tendons (red box; arrowheads). Scale bars 1 µm. 3D reconstruction of SBF-SEM data from (C) WT and (D) Mmp14 KO tendon cells. Brown, cytoplasm. Purple/blue, nucleus. Green, internal collagen fibril compartments. Yellow circles, fibril ends. Lime green (in WT only), Golgi apparatus. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 00610. 7554/eLife. 09345. 007Figure 2—figure supplement 2. Schematic showing looping of collagen fibrils in recessed fibripositors. Diagrammatic representation of a recessed fibripositor with looping of a collagen fibril. Not drawn to scale. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 00710. 7554/eLife. 09345. 008Video 1. Step-through video generated from SBF-SEM images of E17. 5 embryonic WT mouse-tail tendon. z-depth is 100 µm. Scale bar 2 µm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 00810. 7554/eLife. 09345. 009Video 2. Step-through video generated from SBF-SEM images of E17. 5 embryonic Mmp14 KO mouse-tail tendon. z-depth is 100 µm. Scale bar 2 µm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 009 MMP14 can activate proMMP2 (Sato et al., 1994) and proMMP13 (Knauper et al., 1996). Also, MMP2 inhibition blocked uptake and subsequent intracellular digestion of collagen fibrils in periosteal tissue explants (Creemers et al., 1998). However, EM analysis of embryonic Mmp2 KO and Mmp13 KO tail tendons showed no obvious changes to fibripositor occurrence (Figure 3—figure supplement 1). We used SBF-SEM to quantitate the numbers and mean lengths of collagen fibrils in embryonic (E15. 5) WT and Mmp14 KO tendon, using methods described previously (Starborg et al., 2013). WT tendon contained numerous fibril tips and short fibrils (Figure 3A). In contrast, fibril tips were less frequent in KO tendon (Figure 3A). We then calculated the mean length of fibrils based on the relative frequency of tips-to-shaft numbers. The average fibril length in E15. 5 WT tendon was 16 ± 3 μm whereas that in an age-matched and anatomical-site-matched Mmp14 KO tendon was 38 ± 6 μm (Figure 3B). At E15. 5, the tendon cross-sectional area and the FVF in WT and KO samples were not significantly different (Figure 3C, D). Therefore, given the same transverse area occupied by collagen fibrils in WT and KO tissue, the difference in collagen mean fibril length equates to a ∼2. 5-fold reduction in fibril number in KO tendon. Analysis at E16. 5 confirmed that fibrils were, on average, shorter in WT tendon than KO tendon (mean length 50 ± 9 μm) compared with 111 ± 26 μm (n = 668 WT fibrils, 683 KO fibrils, respectively, each tracked over 10 μm). 10. 7554/eLife. 09345. 010Figure 3. Deficiency in MMP14 activity results in fewer collagen fibrils. (A) 10 µm-deep (z-axis) slices of 3D reconstructions of SBF-SEM data taken from of WT and Mmp14 KO embryonic tendons at E15. 5 showing collagen fibrils (blue) with a tip (marked by asterisks) found within the volume. Purple fibrils passed through the volume and so did not have tips in the reconstruction. Scale bars 500 nm. (B) Quantification of mean fibril length based on the number of tips identified shows that E15. 5 WT fibrils are shorter than fibrils in age- and anatomical position-matched tail tendons from KO mice (308 and 266 fibrils tracked, respectively). (C) Tendon cross-sectional area and (D) FVF are not different at E15. 5 KO tendons. (E, F) Electron microscopy of tendon-like constructs cultured in the presence of MMP inhibitor GM6001 (10 µM in 0. 1% DMSO) show increased number of recessed fibripositors (arrowheads) compared to vehicle control. Scale bars 1 µm. (G) Increase in calculated mean fibril length in GM6001-treated tendon-like constructs. Bars show SEM. *p < 0. 05, †p > 0. 05 (t-tests). DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 01010. 7554/eLife. 09345. 011Figure 3—figure supplement 1. Embryonic tendons deficient in Mmp2 or Mmp13 do not have overt tendon phenotypes. Electron microscopy images of embryonic tendons from (A) WT, (B) Mmp2 KO, and (C) Mmp13 KO mice. Arrowheads indicate the locations of fibripositors. Scale bars 1 µm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 01110. 7554/eLife. 09345. 012Figure 3—figure supplement 2. Deficiency in MMP14 activity results in fewer collagen fibrils tips at P0. (A) 3D reconstruction from SBF-SEM analysis of P0 WT and Mmp14 KO tendons. Scale bars 2 µm. (B) Estimated mean fibril length 645 ± 14 µm WT and 674 ± 24 µm Mmp14 KO shows that the absence of MMP14 does not affect fibril length at birth. Bars show SEM. †p > 0. 05 (t-test). DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 01210. 7554/eLife. 09345. 013Figure 3—figure supplement 3. Quantitative analysis of Col-r/r embryonic tendons. Measurement of tendon cross-sectional area (A) and collagen fibril diameter (B) shows no significant difference between WT and Col-r/r mice at E15. 5. †p > 0. 05 (t-tests). (C) Quantitation of fibril-containing carriers in WT, Mmp14 KO, and Col-r/r embryonic tenocytes. *p < 0. 05 (one-way ANOVA). Bars show SEM. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 013 MMP14 has non-proteolytic activities (Mori et al., 2013). Therefore, we treated cells cultured in 3D tendon-like constructs (Kapacee et al., 2008) with the broad-spectrum MMP inhibitor GM6001 and performed TEM. Previous studies had confirmed that GM6001 was an effective inhibitor of MMPs in the tendon (Kalson et al., 2013). The addition of GM6001 to tendon-like constructs recapitulated the fibripositor phenotype of the Mmp14-deficient mouse (Figure 3E, F). This indicated that the catalytic activity of MMP14 is required for normal collagen fibril transport at fibripositors. Estimation of collagen mean fibril lengths in the constructs showed that inhibition of MMPs resulted in increased mean fibril length (Figure 3G), as was observed in embryonic Mmp14 KO tendon. SBF-SEM analysis showed that the mean length of fibrils in WT and Mmp14 KO was the same in P0 tendons (Figure 3—figure supplement 2). This was in contrast to what was seen in embryonic tendon in which the fibrils were longer in KO tendons (Figure 3). Therefore, the more abundant but shorter fibrils in WT tendon grow in length during later embryonic development so that at P0 the fibrils are of equal mean length but there are more fibrils in the WT tendon than in Mmp14 KO tendon. The Col-r/r mouse carries a mutation in the MMP ¾-¼ cleavage site in the triple helical domain of the α1 chain of type I collagen (Liu et al., 1995) that renders both the α1 (I) and α2 (I) chains resistant to cleavage by MMPs (Wu et al., 1990). Therefore, we were interested to compare collagen fibril transport in Col-r/r and Mmp14 KO mice. In contrast to Mmp14 KO, there were no significant differences in tendon size (Figure 3—figure supplement 3A) and collagen fibril diameter (Figure 3—figure supplement 3B) between embryonic (E15. 5) WT and Col-r/r mice. Analysis of the frequency of fibricarrier profiles per nucleus (using methods used previously [Canty et al., 2006]) showed that Mmp14 KO and Col-r/r tenocytes have significantly more fibripositor profiles than WT, with Mmp14 KO cells having the most (Figure 3—figure supplement 3C). SBF-SEM analysis showed that Col-r/r tenocytes contained conspicuous recessed fibripositors, as seen in the Mmp14 KO samples (Figure 2C and Video 3). Electron dense vacuoles were also present, which appeared to contain fibrillar structures in various stages of decomposition (Figure 2C, white arrows). Such compartments were rarely seen in WT or Mmp14 KO samples. 10. 7554/eLife. 09345. 014Video 3. Step-through video generated from SBF-SEM images of E17. 5 embryonic Col-r/r mouse-tail tendon. z-depth is 100 µm. Scale bar 2 µm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 014 We wanted to study the requirement of MMP14 on the stage 1 to stage 2 transition in the tendon but were unable to do so because the global Mmp14 KO mice were distressed shortly after birth. Therefore, we generated a tendon-specific Mmp14 KO mouse by crossing Scleraxis-Cre (Scx-Cre) mice with Mmp14 lox/lox mice (Figure 4—figure supplement 1). We examined the tendons at P0 by TEM and confirmed that the Scx-Cre: : Mmp14 lox/lox tendons also contained multiple fibripositors with multiple fibrils, a similar phenotype observed for global Mmp14 KO mice (Figure 4A, B). At birth the mice appear normal, however, the Scx-Cre: : Mmp14 lox/lox mice exhibited a clear limb phenotype ∼1 week after birth, with dorsiflexion of the fore and hind paws (Figure 4C, D, arrowhead) and a dome-shaped skull (Figure 4D, arrow). There was no apparent impairment in movement of tail or back muscles as observed in Scx-null mice (Murchison et al., 2007). Difference in size was apparent from 3 weeks postnatal concurrent with hip dysplasia and reduced bone density (Figure 4—figure supplement 2A), overgrowth of soft tissues in the paws (Figure 4E) and severe dorsiflexion of paws which was particularly obvious in the hind paws (Figure 4F). Smaller tendon (tail and Achilles) size and weaker bones were also observed in 7-week-old Scx-Cre: : Mmp14 lox/lox mice. The mice became distressed from 7 weeks therefore analyses were performed no later than 7 weeks postnatal. Morphometric analysis of x-rays confirmed that Scx-Cre: : Mmp14 lox/lox mice had significantly shorter cranium length and stunted skeletal growth (Figure 4—figure supplement 2B). 10. 7554/eLife. 09345. 015Figure 4. Scx-Cre: : Mmp14 lox/lox mice have limb and skeletal deformities. Tail tendons from littermates at P0 from (A) WT and (B) Scx-Cre: : Mmp14 lox/lox mice show Mmp14-null tendons have multiple fibripositors-containing multiple fibrils (red box) than fibripositors in WT tendons (blue box). Black arrowhead, recessed fibripositor (electron lucent) -containing collagen fibrils. Scale bars 500 nm. (C) Control pups at P8 showed normal limb development but (D) Scx-Cre: : Mmp14 lox/lox littermates show dorsiflexion of their limbs (arrowhead) and dome-shaped skull (arrow). Adult (7 week old) Scx-Cre: : Mmp14 lox/lox mice have (E) enlarged paws (open arrow) and (F) extreme dorsiflexion of hind limbs (arrowhead) compared to control littermates. Scale bars 1 cm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 01510. 7554/eLife. 09345. 016Figure 4—figure supplement 1. Genotyping the Scx-Cre: : Mmp14 lox/lox colony. A typical genotyping result to identify Scx-Cre: : Mmp14 lox/lox mice. Wt ctrl, control wild-type DNA. Het, heterozygous (lox/wt, Cre+). KO, homozygous knockout (lox/lox, Cre+). WT, wild-type (no Cre). Cre+ WT, Cre-expressing WT (wt/wt, Cre+) used as controls. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 01610. 7554/eLife. 09345. 017Figure 4—figure supplement 2. Adult Scx-Cre: : Mmp14 lox/lox mice have skeletal deformities. (A) Representative x-ray shows adult (7 weeks old) Scx-Cre: : Mmp14 lox/lox mice are smaller than control littermates and have reduced bone density. White arrow indicates hip dysplasia. Scale bars 1 cm. (B) 7-week-old Scx-Cre: : Mmp14 lox/lox mice have shorter cranium length, smaller pelvis, and shorter long bones. Bars show SEM. *p = 0. 0105; ***p < 0. 001 (t-tests; 12 control, 8 Scx-Cre: : Mmp14 lox/lox). DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 017 EM analysis of adult (7 weeks old) WT tendons showed the typical bimodal distribution of collagen fibril diameters (Figure 5A). Cells lacked fibripositors and were stellate in cross section (Figure 5—figure supplement 1A). In contrast, the tendons of Scx-Cre: : Mmp14 lox/lox mice contained a unimodal distribution of small diameter fibrils (Figure 5B), and the cells were engorged with fibrils in membrane-bound compartments (Figure 5—figure supplement 1B). The inclusion of many fibrils into compartments made it difficult to delineate the cell–matrix interface. In comparison, cells in Col-r/r tendons were similar in appearance to those in WT tendons (Figure 5—figure supplement 1C) and the ECM contained fibrils with a broad bimodal distribution of diameters (Figure 5C). 10. 7554/eLife. 09345. 018Figure 5. Deficiency in MMP14 activity inhibits bimodal fibril diameter distribution in tendons from adult mice. Tail tendons from 7 week-old (A) WT, (B) Scx-Cre: : Mmp14 lox/lox, and (C) Col-r/r mice. Larger diameter fibrils can be observed in the ECM of WT and Col-r/r postnatal tendons but only narrow diameter fibrils are observed in Mmp14-deficient postnatal tendons. Scale bars 500 nm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 01810. 7554/eLife. 09345. 019Figure 5—figure supplement 1. Cleavage of the ¾-¼ collagen-I site is not required for release of fibrils in tendons from adult mice. Electron microscopy images of tendons from 7-week-old (A) WT, (B) Scx-Cre: : Mmp14 lox/lox, and (C) Col-r/r mice. Larger diameter fibrils occur in the ECM of WT and Col-r/r postnatal tendons but only narrow diameter fibrils occur in Mmp14-deficient postnatal tendons. Scale bars 2 µm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 01910. 7554/eLife. 09345. 020Figure 5—figure supplement 2. Immuno-electron microscopy of Scx-Cre: : Mmp14 lox/lox tendon. (A) WT and (B) Scx-Cre: : Mmp14 lox/lox tail tendon from P7 (7 days) mice were labeled with anti-MMP14 antibody. (C) Scx-Cre: : Mmp14 lox/lox tendon labeled with anti-collagen-I antibody. Scale bars 500 nm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 020 ImmunoEM of P7 (7 days postnatal) WT tendon using an anti-MMP14 antibody showed labeling of intracellular compartments without labeling the ECM or plasma membranes in contact with extracellular collagen fibrils (Figure 5—figure supplement 2A). As expected, Mmp14-null tendons were negative for MMP14 labeling (Figure 5—figure supplement 2B). Labeling using an anti-type I collagen antibody confirmed that the fibrils within the recessed fibripositors of Scx-Cre: : Mmp14 lox/lox tendons contained type I collagen (Figure 5—figure supplement 2C). To investigate why collagen fibrils were retained in fibripositors in Mmp14-null tendons, we used mass spectrometry LS/MS–MS to perform an unbiased comparison of proteins in Mmp14-deficient and WT Achilles tendons. Care was taken to minimize muscle contamination and to remove as much associated loose connective tissue as possible. Although not quantitative, the analysis identified specific macromolecules that appear to be more abundant in Scx-Cre: : Mmp14 lox/lox and global Mmp14 KO tendons than in WT (Supplementary file 1). Peptides from FN were consistently more abundant in Mmp14-deficient samples. We identified a unique peptide from FN that was found in WT tendon in vivo and which contained an additional alanine residue at its N-terminus in Mmp14-deficient tendons (Figure 6A), suggesting that MMP14 is responsible for cleaving FN between Ala (1078) and Thr (1079). This was confirmed by LS/MS–MS analysis of recombinant human FN treated with recombinant human MMP14 prior to digestion with trypsin (data not shown). Immunofluorescence analysis at E15. 5 showed accumulation of FN in Scx-Cre: : Mmp14 lox/lox tendons compared to WT tendons and the levels of FN appeared to progressively accumulate in KO tendons at P0 and P10 (Figure 6B). The LS-MS/MS analyses also identified periostin and integrins including α11β1 (Supplementary file 1). Analysis of periostin in tendons showed similar intensities at E15. 5 and P0 but was increased in the tendon epithelium at P10 Scx-Cre: : Mmp14 lox/lox mice compared to WT mice (Figure 6—figure supplement 1A). Subsequent western blot analysis of P7 mice confirmed that levels of FN were higher in Scx-Cre: : Mmp14 lox/lox tendons compared to WT littermates (Figure 6C, D). We stripped and re-probed the blot for periostin detection and confirmed that it was not accumulated to the extent FN was in Scx-Cre: : Mmp14 lox/lox tendons (Figure 6—figure supplement 1B). 10. 7554/eLife. 09345. 021Figure 6. Elevated FN in Mmp14-deficient tendons. (A) Sequence of a unique semi-tryptic peptide of FN identified in neonatal (P7-10) WT tendon and the sequence of the corresponding peptide from Mmp14-deficient tendons without the additional Ala (1078) -Thr (1079) cleavage. (B) Immunofluorescence analysis of FN in tendons of WT and Scx-Cre: : Mmp14 lox/lox mice at E15. 5, P0, and P10 of development. Scale bars 200 µm. (C) Western blot analysis of P7 WT and Scx-Cre: : Mmp14 lox/lox tendons show elevated FN in Mmp14-deficient tendons. (D) Ponceau S-stained membrane shows equivalent extractability of WT and Scx-Cre: : Mmp14 lox/lox tendons. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 02110. 7554/eLife. 09345. 022Figure 6—figure supplement 1. Elevated periostin levels only in postnatal Scx-Cre: : Mmp14 lox/lox tendons. (A) Immunofluorescence analysis of periostin in tendons of WT and Scx-Cre: : Mmp14 lox/lox mice at E15. 5, P0, and P10 of development show accumulation of periostin in epithelium (white arrows) of Scx-Cre: : Mmp14 lox/lox tendons. Scale bars 200 µm. (B) Western blot analysis of P7 WT and Scx-Cre: : Mmp14 lox/lox Achilles tendons show similar levels of periostin in Mmp14-deficient tendons. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 02210. 7554/eLife. 09345. 023Figure 6—figure supplement 2. Exogenous FN induces recessed fibripositors in tendon-like constructs. Transmission electron microscopy of tendon-like constructs in the absence (A) and presence (B) of 200-µg/ml human plasma fibronectin. Arrowheads show recessed fibripositors, which were abundant in the treated constructs. Scale bars 500 nm. DOI: http: //dx. doi. org/10. 7554/eLife. 09345. 023 Next, we wanted to determine if elevated levels of FN might account for the fibripositor phenotype and retention of collagen fibrils at the cell surface. Thus, we formed tendon-like constructs in the presence of 200 µg/ml exogenous human plasma FN. The constructs formed within ∼10 days as previously described (Kapacee et al., 2008). TEM of the constructs showed pronounced fibripositors in cells incubated in exogenous FN (Figure 6—figure supplement 2). We show here that MMP14 is essential for the stage 1–stage 2 transition of tendon development (which occurs around birth in the mouse) by catalyzing the release of collagen fibrils from fibripositors. In WT mouse tendons, fibripositors disappear and collagen fibrils are released to the ECM soon after birth (marking the end of stage 1), and the fibrils grow in diameter and length (marking the start of stage 2) (Kalson et al., 2015). In the absence of MMP14, the fibrils are retained within fibripositors and the number of collagen fibrils formed during stage 1 is reduced. As a result, tendons in Mmp14-deficient mice are thinner compared to WT. Although MMP14 is capable of cleaving type I collagen at the ¾-¼ helical site, we show that cleavage at this site is not required for tendon development. Interestingly, FN accumulates in Mmp14-deficient tendons. Thus, we propose that the ability of MMP14 to cleave macromolecules other than type I collagen is essential for releasing collagen fibrils from fibripositors. Collagen fibrils are assembled on the surface of embryonic tenocytes and pulled into fibripositors by a mechanism powered by non-muscle myosin II (Kalson et al., 2013). The absence of fibripositors in stage 2 shows that fibrils are ‘released’ from the plasma membrane for delivery to the ECM (Kalson et al., 2015). We propose that collagen fibrillogenesis in embryonic tendon occurs via an ‘APR’ mechanism of collagen ‘attachment’ to the cell surface, non-muscle myosin II-powered ‘pulling’ on fibrils into fibripositors, and MMP14-mediated ‘release’ of the fibrils to the ECM. Mmp14-null mice had thinner tendons than WT mice. SBF-SEM analyses showed similar cell numbers in embryonic WT and null mouse tendons; therefore, we excluded the possibility that delayed development was the cause. However, Mmp14-null tendons had fewer collagen fibrils. As shown previously, the lateral size of the tendon is established during embryonic development when embryonic tenocytes assemble a finite number of collagen fibrils at fibripositors (Kalson et al., 2015). The fibrils are released after birth and subsequently grow in length and diameter in a process of matrix expansion. Therefore, the fewer fibrils in Mmp14-deficient tendons appear to be a direct result of the inability of the cells to release collagen fibrils to the ECM before a new cycle of fibril assembly can begin. In the presence of continued collagen synthesis (albeit at a reduced rate [Figure 1—figure supplement 1D]), the existing fibrils continue to grow in length and diameter at the expense of nucleation of new fibrils. At P0 and soon after birth, the fibrils in WT tendon grow in length to equal the length of fibrils in Mmp14 KO tendons. Tendon size was normal in Col-r/r embryos; therefore, the absence of type I collagen cleavage was not the cause of reduced fibril number in Mmp14-deficient tendons. However, tenocytes in embryonic Col-r/r tendons contained electron-dense vacuoles, which were morphologically similar to those previously observed (Beertsen et al., 1978; Everts et al., 1996). Therefore, although type I collagen is cleaved at the ¾-¼ site during embryonic development, cleavage is not essential for tendon development. While the Col-r/r mutation renders the triple helix resistant to cleavage by MMPs, rodent MMP13 recognizes an additional cleavage site C-terminal to the N-telopeptide crosslink (Krane et al., 1996). Liu et al. (1995) reported that the MMP13 cleavage site in type I collagen permits normal remodeling during development and early postnatal life, but that cleavage at the ¾-¼ site is needed for subsequent remodeling and accounts for the observed progressive marked skin fibrosis in the Col-r/r. Our data agree with these conclusions and show that MMP13 (and MMP2) is not essential for tendon development. The fact that degradative vacuoles are rare in embryonic Mmp14 KO tenocytes and that fibril numbers were reduced and fibril lengths are greater in Mmp14 KO cells, suggests that the vacuoles might be part of a mechanism to regulate fibril number and/or length. Two proteins, FN and periostin, stood out in the LC-MS/MS comparison of WT and Mmp14-null tendons as proteins that could help to explain the Mmp14-null tendon phenotype (Supplementary file 1 shows number of peptides from periostin and FN were over-represented in Mmp14 KO tendon). Periostin is a member of the matricellular family of secreted proteins that modulate cell–ECM interactions (Sage and Bornstein, 1991; Murphy-Ullrich and Sage, 2014). Periostin is highly expressed by epithelial cells, is bound by αvβ3 and αvβ5 integrins, is upregulated in epithelial tumors to support adhesion and migration (Gillan et al., 2002; Yuyama et al., 2002; Liu and Du, 2015), and is a prognostic marker for TH2-driven asthma (Parulekar et al., 2014) and lung fibrosis (Amara et al., 2015). Additional studies have shown that periostin supports tendon formation in an ectopic mouse model of the development of tenogenic tissue (Noack et al., 2014). Evidence also suggests that periostin interacts with type I collagen to regulate collagen fibrillogenesis (Noack et al., 2014). It has also been reported that periostin deficiency might cause collagen fibril disorganization and affect the distribution of FN (Tabata et al., 2014). We showed that periostin was increased in the tendon epithelium (Taylor et al., 2011) that surrounds the body of the tendon, in P10 Scx-Cre: : Mmp14 lox/lox mice compared to WT mice (Figure 6—figure supplement 1A). Periostin can be cleaved by MMP14 in vitro (Stegemann et al., 2013) and therefore its accumulation in the epithelium could be a direct result of substrate accumulation. It is also possible that the elevated levels seen in the epithelium are an indirect result of MMP14 deficiency in the fibrous core of the tendon. We observed elevated levels of FN in Mmp14 KO tendon, as shown by LC-MS/MS and by immunofluorescence. MMP14 has been shown to cleave several macromolecules in vitro including FN (d' Ortho et al., 1997; Ohuchi et al., 1997; Tam et al., 2004; Butler and Overall, 2007); therefore, the accumulation of FN in Mmp14-deficient tendon might be a direct result of the absence of MMP14. We observed a potential cleavage site between Ala (1078) and Thr (1079) in FN that occurred in WT but not in Scx-Cre: : Mmp14 lox/lox tendon. FN is a core component of extracellular matrices (Mao and Schwarzbauer, 2005) and has an important role in development (George et al., 1993) and wound healing (Sakai et al., 2001). Mice lacking FN are embryonic lethal with defects in mesoderm formation (George et al., 1993). Furthermore, FN co-distributes with type I and III collagen (Fleischmajer and Timpl, 1984). To understand if the presence of elevated levels of FN could explain the fibripositor phenotype, we incubated tendon-like constructs with exogenous FN. This resulted in a profound increase in appearance of fibripositors. Taking the EM, LC-MS/MS, and tendon-construct data together, we propose that FN forms a ‘molecular bridge’ between the cell and the collagen fibril. Thus, cleavage of the bridge and removal of fibripositors triggers the onset of stage 2 of tendon development and subsequent expansion of the matrix. An unexpected observation was the effect on skeletal size of deleting MMP14 from tendon. The shortened long bones in Scx-Cre: : Mmp14 lox/lox mice suggests important consequences of tendon development on skeletal growth. LC-MS/MS analyses showed under-representation of decorin, COMP, PCOLCE, and TNXB in Mmp14-null tendons. These proteins are directly involved in regulating collagen fibril size and shape; mice lacking decorin have fibrils with irregular outlines (Danielson et al., 1997), COMP can act as a catalyst for collagen fibril formation (Halasz et al., 2007), PCOLCE enhances the cleavage of procollagen to collagen (Takahara et al., 1994) and mice lacking TNXB have reduced numbers of collagen fibrils (Mao et al., 2002). In contrast, several proteins were over-represented in Mmp14-deficient tendons. These included filamin A, which has functions in cell–ECM adhesion (Nakamura et al., 2011) and mechanotransduction (Jahed et al., 2014). As tendons are predominately ECM, changes in ECM composition and cell–ECM interactions are likely to have profound effects on cell signaling (e. g., ECM growth factor presentation) as well as the growth and mechanical properties of tendon leading to changes in musculoskeletal development. Finally, the tendons, cartilage, muscle, and bone are peripheral circadian clocks, each with their unique circadian transcriptome (see [Yeung et al., 2014a] and reviewed by Dudek and Meng (2014) ). Therefore, changes in the organization and mechanical properties of the tendon might affect its circadian entrainment and that of adjacent musculoskeletal tissues. The care and use of all mice in this study was carried out in accordance with UK Home Office regulations, UK Animals (Scientific Procedures) Act of 1986 under the UK Home Office licence (PPL 40/3485). All animals were sacrificed by a Schedule 1 procedure by trained personnel. Mmp14 KO mice were as described previously (Zhou et al., 2000). To generate mice in which Mmp14 is ablated in tendon-lineage cells, we crossed mice-expressing Cre recombinase under the control of Scleraxis (Scx-Cre; C57BL/6) (Blitz et al., 2013) with mice carrying the floxed exons (exons 2 to 4) of the Mmp14 gene (Mmp14 lox/lox; C57BL/6) (Zigrino et al., 2012). Mmp13 KO embryos were a generous gift from Zena Werb (Stickens et al., 2004). Mmp2 heterozygous mice were imported from RIKEN BioResource Center (GelAKO/RBRC00398; C57) (Itoh et al., 1997) and bred to homozygosity. Col-r/r mice were imported from Jackson Laboratory (B6; 129S4-Col1a1tm1Jae/J) (Liu et al., 1995). X-ray analyses were performed as described previously (Yeung et al., 2014b). The methods used were as described previously (Kalson et al., 2010). Tendon (from tail) diameters were measured from digital photographs. The diameter, d, was then used to calculate transverse area according to the formula πd2/4. This assumed a circular transverse shape as used in mechanical testing of tissue engineered ligament (Hairfield-Stein et al., 2007). An average of three diameter measurements was recorded for each tendon. The original contour length of tendons was measured from a digital photograph of the mounted construct. A tare load of 10 mN was applied at the start of the tensile test to fully straighten the tendon. The length at failure was determined from the Instron test (giving change in length LΔ). The tendons were tested to failure with a strain rate of 5 mm per minute (equivalent to approximately 1% strain per second). A minimum of 3 tail tendons was examined for each experiment. The tendons were prepared for TEM and SBF-SEM as described (Starborg et al., 2013), with care being taken to maintain the length and tension during fixation. Sections (70-nm thick) were examined for TEM using an FEI Tecnai 12 instrument fitted with a 2k × 2k-cooled CCD camera (F214A, Tietz Video and Image Processing Systems, Gauting, Germany). Serial section electron tomography was completed as described using semi-thick (300 nm) serial sections were collected on formvar-coated copper slot grids. Orthogonal tilt series were then acquired on a FEI Tecnai Polara TEM operated at 300 kV (Kalson et al., 2013). Tomograms were generated and contours modeled in IMOD (Kremer et al., 1996). The methods used for SBF-SEM were as described (Starborg et al., 2013) using a Gatan 3View microtome within an FEI Quanta 250 scanning microscope. Cell number measurements were made on 3 separate SBF-SEM samples for WT and Mmp14 KO tail tendons at P0. The volume of the tendon tissue in each SBF-SEM 3D reconstruction was calculated and all the cells in the volume were reconstructed using IMOD. Each cell nucleus contained within the reconstruction was identified and counted. Cells per 1000 μm3 of tissue were calculated to allow comparison between samples. ImmunoEM was performed as previously described using high-pressure freezing and freeze substitution into LR White resin (Canty et al., 2004). A rabbit anti-Collagen-I antibody (T40777R; Meridian Life Science, Inc.) and a mouse anti-MMP14 antibody (MAB3328; Merk Millipore) were used. Cleanly dissected neonatal (P7-10) mouse Achilles tendons were snap frozen in liquid nitrogen and disrupted in 0. 1 M Tris, pH 7. 5 using a B Braun Mikro-dismembrator S (2 × 90 s, 2000 rpm). Tissue samples were digested with trypsin (12. 5 ng/µl; Sigma) overnight at 37°C in 25 mM ammonium bicarbonate (pH 7. 5). Human rhFurin (2 ng/ml) in 100 µl activation buffer (50 mM Tris-HCl, 1 mM CaCl2,0. 5% Brij-35, pH 9) was added to 4 μl human rhProMMP14 (0. 37 μg/μl) and incubated for 1. 5 hr at 37°C. Recombinant human FN (1 µg/ml) was incubated with activated rhMMP14 (1 ng/ml) at 37°C for 1 hr. For gel-top analysis, MMP14-treated FN was briefly separated by electrophoresis under reducing conditions. A gel top band was excised from the stained gel and processed using in-gel tryptic digestion. For in-gel tryptic digestion, proteins were excised from SDS-PAGE gels and dehydrated using acetonitrile followed by vacuum centrifugation. Dried gel pieces were reduced with 10 mM dithiothreitol and alkylated with 55 mM iodoacetamide. Gel pieces were then washed alternately with 25 mM ammonium bicarbonate followed by acetonitrile and dried by vacuum centrifugation. Samples were digested with trypsin, as above. Digested samples were analyzed by LC-MS/MS using an UltiMate 3000 Rapid Separation LC (RSLC, Dionex Corporation, Sunnyvale, CA) coupled to an Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA) mass spectrometer. Peptide mixtures were separated using a gradient from 92% A (0. 1% FA in water) and 8% B (0. 1% FA in acetonitrile) to 33% B, in 44 min at 300 nl/min, using a 250 mm × 75 μm i. d. 1. 7 μM BEH C18, analytical column (Waters). Peptides were selected for fragmentation automatically by data-dependent analysis. Data produced were searched using Mascot (Matrix Science UK), against uniprot with taxonomy of Mus musculus selected. Cysteine carbamidomethylation was selected as a fixed modification, and lysine and proline oxidation included as variable modifications, for all enzymes. For trypsin, methionine oxidation was additionally included as a variable modification and the enzyme selected as ‘semi-trypsin’. Data were validated using Scaffold (Proteome Software, Portland, OR). Mouse Achilles tendons (at P7) were dissected clean of contaminating the muscle, snap frozen in liquid nitrogen and disrupted using a B Braun Mikro-dismembrator S (2 × 90 s, 2000 rpm). Proteins were extracted directly into RIPA buffer (50 mM Tris, pH 7. 6,150 mM NaCl, 0. 1% SDS, 1 mM EDTA and 1% NP-40) containing EDTA-free protease inhibitor cocktail (Roche) and quantified using a BCA assay. Samples (100 µg) were reduced and analyzed by western blotting and densitometry. Western blots were stripped with 2% SDS, 62. 5 mM Tris HCl pH 6. 8 and 100 mM 2-mercaptoethanol for 50 min at 50°C. Fibronectin (FN) was detected using a rabbit polyclonal antibody (ab2413; Abcam), and periostin was detected using a goat polyclonal antibody (AF2955; R&D Systems). From WT and Scx-Cre: : Mmp14 lox/lox mice, whole hind limbs from E15. 5 embryos, lower hind limbs without the skin from P0 pups and dissected Achilles tendons from P10 pups were cryo-preserved in OCT-embedding matrix (Thermo Scientific). Longitudinal sections of 8-µm thickness were fixed with 4% paraformaldehyde in PBS for 15 min, permeabilized with 0. 2% Triton X-100 in PBS for 10 min and then blocked with 2% BSA in PBS for 1 hr. FN was detected using a rabbit polyclonal antibody (ab23750; Abcam; diluted 1: 500), and periostin was detected using a goat polyclonal antibody (ab14041; Abcam; diluted 1: 500). Cy3-conjugated secondary antibodies (Invitrogen) were used and sections were mounted using Vector Shield containing DAPI (Vector Laboratories). Fluorescent images were taken using a digital camera attached to an Olympus BX51 and captured using MetaVue imaging software (Molecular Devices). P7 tail tendons were labeled with 14C-proline for 1 hr and separate extracellular and intracellular extracts prepared as described (Canty et al., 2004). The intracellular extract was analyzed by electrophoresis using 4–12% pre-cast Bis-Tris gels and MES running buffer. The gels were divided and the top half of the gel (>70 kDa) fixed, dried, and analyzed by autoradiography to detect 14C-collagen. The bottom half of the gel (<70 kDa) was analyzed by Western blotting with an antibody to β-actin; signal was detected using a CCD camera system. The gels were analyzed by densitometry with the relative band intensities determined by comparison to serial dilutions of an independent sample. | Title: Matrix metalloproteinase 14 is required for fibrous tissue expansion Summary: A scaffold of proteins called the extracellular matrix surrounds each of the cells that make up our organs and tissues. This matrix, which contains fibres made of proteins called collagens, provides the physical support needed to hold organs and tissues together. This support is especially important in the tendons-a tough tissue that connects the muscle to bone-and other 'connective' tissues. An enzyme called MMP14 is able to cut through chains of collagen proteins. It belongs to a family of proteins that are involved in breaking down the extracellular matrix to enable cells to divide and for other important processes in cells. Some cancer cells exploit MMP14 to enable them to leave their tissue of origin and spread around the body. Therefore, when researchers bred mutant mice that lacked MMP14, they expected to see excessive growth of collagen fibres in the connective tissues of the mice. However, these mice actually have extremely thin, fragile connective tissue and die soon after birth. Earlier in 2015, a group of researchers demonstrated that the first stage of tendon development in mice involves the formation of collagen fibres, which are attached to structures that project from tendon cells called fibripositors. Then, soon after the mice are born, the fibripositors disappear and the collagen fibres are released into the extracellular matrix where they grow longer and become thicker. Now, Taylor, Yeung, Kalson et al. -including some of the researchers from the earlier work-have used electron microscopy to investigate how a lack of MMP14 leads to fragile tendons in young mice. The experiments show that MMP14 plays a crucial role in the first stage of tendon development by detaching the collagen fibres from the fibripositors. MMP14 also promotes the formation of new collagen fibres; the tendons of mutant mice that lack MMP14 have fewer collagen fibres than normal mice. Further experiments revealed that the release of collagen fibres from fibripositors does not require MMP14 to cleave the chains of collagen proteins themselves. Instead, it appears that MMP14 cleaves another protein that is associated with the fibres, called fibronectin. Taylor, Yeung, Kalson et al.' s findings show that MMP14 plays an important role in the development of tendons by releasing collagen fibres from fibripositors and promoting the formation of new fibres. The next challenge is to find out how MMP14 regulates the number of collagen fibres in mature tendons and other tissues, and how defects in this enzyme can lead to cancer and other diseases. | 14,119 | 590 | lay_elife | en |
Summarize: Background VBA’s compensation program pays monthly benefits to veterans with service-connected disabilities (injuries or diseases incurred or aggravated while on active military duty), according to the severity of the disability. VBA’s pension program pays monthly benefits to wartime veterans who have low incomes and are permanently and totally disabled for reasons not service-connected. In addition, VBA pays dependency and indemnity compensation to some deceased veterans’ spouses, children, and parents. In fiscal year 2001, VBA paid over $20 billion in disability compensation to about 2.3 million veterans and over 300,000 survivors. VBA also paid over $3 billion in pensions to over 600,000 veterans and survivors. Veterans may submit their disability claims to any of VBA’s 57 regional offices, which process these claims in accordance with VBA regulations, policies, procedures, and guidance. Regional offices assist veterans in obtaining evidence to support their claims. This assistance includes helping veterans obtain the following documents: records of service to identify when the veteran served, records of medical treatment provided while the veteran was in military service, records of treatment and examinations provided at VA health-care facilities, and records of treatment of the veteran by nonfederal providers. Also, if necessary for decision on a claim, the regional office arranges for the veteran to receive a medical examination or opinion. Once this evidence is collected, VBA makes a rating decision on the claim. Veterans with multiple disabilities receive a single composite rating. For pension claims, VBA determines whether the veteran meets certain criteria. The regional office then notifies the veteran of its decision. In May 2001, the Secretary created the VA Claims Processing Task Force to develop recommendations to improve the compensation and pension claims process and to help VBA improve claims processing timeliness and productivity. The task force observed that the work management system in many VBA regional offices contributed to inefficiency and an increased number of errors. The task force attributed these problems primarily to the broad scope of duties performed by regional office staff—in particular, veterans service representatives (VSR). For example, VSRs were responsible for both collecting evidence to support claims and answering claimants’ inquiries. In October 2001, the task force made short- and medium-term recommendations for improving the claims process and reorganizing regional office operations. In particular, the task force recommended that VBA change its claims processing system to one that utilizes specialized teams. VBA is in the process of implementing many of these recommendations and has established a new claims processing structure that is organized by specific steps in the claims process. For example, regional offices will have teams devoted specifically to claims development, that is, obtaining evidence needed to evaluate claims. VBA’s Key Timeliness Measure Does Not Clearly Reflect Its Performance VBA’s key timeliness measure does not clearly reflect its timeliness in completing claims because it fails to distinguish among its three disability programs—disability compensation, pension, and dependence and indemnity compensation. The programs’ processing times differ, in part because they have different purposes, beneficiaries, eligibility criteria, and evidence requirements to decide each type of claim. Despite these differences, VBA sets an annual performance goal that is an average of all three programs. For the purposes of reporting its performance to the Congress and other stakeholders, VBA adopted one key timeliness measure—the average time to complete decisions on rating-related cases. This measure includes original and reopened disability compensation, pension, and dependency and indemnity compensation claims—in other words, claims for three VBA compensation and pension programs. VBA sets an annual goal for average days to complete rating-related cases in VA’s annual performance plans and subsequently reports its actual timeliness—and whether it met its goal—in VA’s annual performance reports to the Congress. This one measure does not reflect the differences in the timeliness for the three programs. In general, the disability compensation program requires the most evidence and thus these claims generally take longer to complete, as shown in figure 1. While VBA’s average fiscal year 2002 timeliness was 223 days, disability compensation decisions (which represented about 83 percent of total decisions) took almost twice as long to complete as pension decisions. The aggregate measure understated the time required to decide disability compensation claims by 18 days and overstated the time to decide pension claims by 97 days and dependency and indemnity compensation claims by 51 days. Each program has a different claims processing time frame because each has different evidence requirements resulting from their different purposes and eligibility requirements. For example, a major reason why disability compensation claims take longer is that VBA must not only establish that each claimed disability exists, but that each was caused or aggravated by the veteran’s military service. This process requires substantial evidence gathering, with VBA actively assisting the claimant. To prove service- connection, VBA obtains the veteran’s service medical records and may request medical examinations and treatment records from VA medical facilities. In contrast, pension claims do not require evidence that the claimed disabilities were service-connected. Also, veterans aged 65 and older do not have to prove that they are disabled to receive pension benefits as long as they meet the income and military service requirements. VBA Does Not Have Adequate Data to Measure Timeliness of Claims Processing Teams, but Is Making Progress VBA does not yet have adequate data to measure timeliness or set goals for its specialized regional office teams but is making progress in obtaining complete and accurate data. While VBA is in the process of developing performance measures and goals for these teams and has developed a system to report timeliness data, it acknowledges that the quality of its existing timeliness data needs to be improved. Implementation of the task force recommendations to reorganize claims processing requires that VBA measure its performance for its teams. Where teams were once responsible for processing claims from receipt to completion, teams are now responsible for specific phases of the process. With complete and accurate data, VBA will be able to measure the timeliness of the individual teams and, therefore, will be able to hold them accountable for their performance as well as identify processing delays and take corrective actions. VBA expects to be able to obtain more complete and accurate data to measure team performance once it deploys new software applications that should enable it to consistently capture data for all cases and will rely less on manual data entry. VBA expects these applications to be fully deployed by October 2003. The task force recommended that regional office Veterans Service Centers (VSC), which process compensation and pension claims, be reorganized into specialized teams. The task force identified six types of teams—triage, pre-determination, rating, post-determination, appeals, and public contact—based on different phases of the claims process. From February through April 2002, VBA piloted its CPI initiative, which included reorganizing regional offices’ VSCs into specialized teams at four regional offices. The CPI task team noted that processing teams needed clearly defined and reasonable performance expectations and recommended timeliness measures for each team, as shown in table 1. VBA began to implement the CPI model at its other regional offices in July 2002. VBA has implemented an inventory management system (IMS) that allows it to measure and report team timeliness, nationally and at the regional office level. This system should provide VBA with the necessary data to develop annual performance goals, which can be used to hold itself and its regional offices accountable for improving timeliness. IMS should also provide useful data to assist VBA management with identifying problems in specific regional offices and allowing regional office management to identify problems with specific teams for further analysis and corrective actions. However, VBA acknowledges that its IMS reports are not as useful as they can be, because IMS receives incomplete data from an existing VBA system—the Claims Automated Processing System (CAPS). Not all regional offices are fully using CAPS; thus, CAPS data that are sent to IMS are incomplete. CAPS was not being used to collect timeliness data for all cases; rather, it was used to provide regional office staff with information on the status of cases expected to take more than 30 days to process. In order to provide a short-term improvement in the completeness of IMS data, in May 2001 VBA instructed regional offices to ensure that certain data were consistently entered into CAPS; for example, dates when evidence was requested and received. In May 2002, VBA instructed regional offices to report on how fully they use CAPS and to provide estimated timeframes for complete compliance with CAPS data entry requirements. As of August 2002, VBA reported that about 81 percent of its pending cases had records in CAPS. According to VBA officials, as the regional offices implement new software applications, the ability of IMS to provide complete and accurate timeliness reports is expected to improve. For example, Share, the new claims establishment application, will automatically input data on a case into other applications, including CAPS. This will help ensure more complete and consistent data in the short term, because there will be a CAPS record for each case. Eventually, the Modern Award Processing– Development (MAP-D) application will replace CAPS as a source of timeliness data for IMS. MAP-D will, according to VBA officials, contain records for all cases and will reduce the amount of manual data entry required, thus reducing the potential for data input errors. VBA plans to have all regional offices using Share and MAP-D by October 2003. Conclusions VBA has chosen to use one aggregate performance measure for timeliness for its disability compensation, pension, and dependency and indemnity compensation programs. Such a measure does not reflect VBA’s performance for programs with different purposes, beneficiaries, and claims processing requirements. In particular, VBA’s timeliness in deciding disability compensation claims is assessed under a measure that also covers pension and dependency and indemnity compensation claims, which take much less time. Consequently, the aggregate measure can make the processing time for VBA’s largest and most time-consuming workload look better than it really is. As long as VBA uses an aggregate timeliness measure, it will not be able to clearly demonstrate to the Congress, top VA management, and claimants how well it is meeting its objectives to serve disabled veterans and their families. VBA’s reorganization of its regional office compensation and pension claims processing operations into specialized teams underscores the need for complete and accurate data on the timeliness of the phases of the claims process. VBA does not yet have adequate data for timeliness measurement purposes but is making progress in ensuring that it does. Once VBA has deployed its new claims processing software applications at all of its regional offices, it expects to be able to better measure the timeliness of its specialized teams, provide baselines for future comparisons, quantify team performance goals, and identify problems needing corrective action. In this way, local and team-specific information can be used to hold regional offices and their specialized teams accountable for improving timeliness. Recommendation We recommend that the Secretary of Veterans Affairs direct the Under Secretary for Benefits to establish separate claims processing timeliness goals for its three main disability programs, incorporate these goals into VA’s strategic plan and annual performance plans, and report its progress in meeting these goals in its annual performance reports. Agency Comments and Our Response In its written comments on a draft of this report (see app. I), VA concurred in principle with our recommendation. VA noted that VBA plans to develop performance measures for each of its programs, as part of VA’s effort to restructure its budget. However, VA believes establishing new goals by program should be deferred until at least fiscal year 2005, because establishing new goals at this time risks obscuring its focus on achieving the Secretary’s 100-day goal by the end of fiscal year 2003. We believe developing timeliness measures for each program would not obscure VBA’s focus on performance improvement, but would provide a more accurate picture of claims processing timeliness, because the new measures would reflect the differences among the three programs. Because VBA already has the necessary data, we believe that it should report timeliness by program for fiscal year 2004 and set goals by program for fiscal year 2005, at the latest. VA also suggested that we based our calculations of average days to complete disability compensation, pension, and dependency and indemnity compensation decisions, as shown in figure 1, on original claims only. We based our calculations on all eight types of claims (known as end products) that VBA uses to calculate rating-related timeliness. These end products include both original and reopened claims. As agreed with your offices, unless you publicly announce its contents earlier, we plan no further distribution of this report until 1 day after its issue date. At that time, we will send copies of this report to the Secretary of the Department of Veterans Affairs, appropriate congressional committees, and other interested parties. We will also make copies of this report available to others on request. The report will also be available at no charge on GAO’s Web site at http://www.gao.gov. If you or your staff have any questions regarding this report, please call me at (202) 512-7101 or Irene Chu, Assistant Director, at (202) 512-7102. In addition to those named, Susan Bernstein, Martin Scire, and Greg Whitney made key contributions to this report. Appendix I: Comments from the Department of Veterans Affairs | Summary: The Chairman and Ranking Minority Member, Senate Committee on Veterans' Affairs, asked GAO to assist the Committee in its oversight of the Veterans Benefits Administration's (VBA) efforts to improve compensation and pension claims processing. As part of this effort, GAO assessed (1) whether VBA's key timeliness measure clearly reflects its performance and (2) whether it has adequate data to measure the timeliness of its newly created specialized claims processing teams. VBA's key claims processing timeliness measure does not clearly reflect how quickly it decides claims by veterans and their families for disability compensation, pension, and dependency and indemnity compensation benefits. Although each program has its own purpose and eligibility requirements, VBA does not set a separate timeliness goal for each in its annual performance plan. This obscures the significant differences in the time required to complete decisions under each program. Fiscal year 2002 timeliness, using VBA's measure, was 223 days; however, disability compensation decisions took significantly longer than decisions under the other two programs. A disability compensation decision requires more evidence, in part because VBA must determine that each claimed disability is related to the veteran's military service. VBA does not yet have adequate data to measure the timeliness of its new specialized regional office claims processing teams but is working to improve its data. VBA's inventory management system, which allows it to report and analyze teams' timeliness, relies on an existing information system that does not provide timeliness data on all cases. VBA is acting to improve the completeness of the data in the existing system. Meanwhile, VBA is deploying new software that it expects should enable it to capture more complete and accurate data. VBA expects to deploy this new software at all regional offices by October 2003. | 3,004 | 397 | gov_report | en |
Write a title and summarize: Blocking Plasmodium falciparum transmission to mosquitoes has been designated a strategic objective in the global agenda of malaria elimination. Transmission is ensured by gametocyte-infected erythrocytes (GIE) that sequester in the bone marrow and at maturation are released into peripheral blood from where they are taken up during a mosquito blood meal. Release into the blood circulation is accompanied by an increase in GIE deformability that allows them to pass through the spleen. Here, we used a microsphere matrix to mimic splenic filtration and investigated the role of cAMP-signalling in regulating GIE deformability. We demonstrated that mature GIE deformability is dependent on reduced cAMP-signalling and on increased phosphodiesterase expression in stage V gametocytes, and that parasite cAMP-dependent kinase activity contributes to the stiffness of immature gametocytes. Importantly, pharmacological agents that raise cAMP levels in transmissible stage V gametocytes render them less deformable and hence less likely to circulate through the spleen. Therefore, phosphodiesterase inhibitors that raise cAMP levels in P. falciparum infected erythrocytes, such as sildenafil, represent new candidate drugs to block transmission of malaria parasites. Recent renewed emphasis on the eradication of malaria has highlighted the need for novel interventions to target the parasite during transmission from the human host to the mosquito. Drug treatments to clear asexual blood stage parasites (that cause pathology) do not kill mature gametocytes and therefore allow transmission to continue [1]. Transmission of malaria parasites relies on the sexual stages, termed gametocytes that circulate in the peripheral blood and are taken up by Anopheles mosquitos during a blood meal. For Plasmodium falciparum, the causative agent of the most severe form of human malaria, gametocyte maturation requires about 10 days and is divided in five morphological stages [2]. During this period, immature gametocyte-infected erythrocytes (GIE) sequester in internal organs such as bone marrow and spleen [3–6]. Sequestration mechanisms of GIE are still unknown, although failure of immature GIE to adhere to endothelial cell lines in vitro [7], and absence on their surface of parasite structures allowing cytoadhesion of asexual stages [8], suggest that GIE-host interactions are unlikely to be mediated by cytoadhesion. Recent evidence rather suggests that GIE biomechanical properties may play an important role in this process [9]. At maturation GIE are released into the blood circulation, where they can persist for several days [10], thus increasing the likelihood of parasites being taken up during a mosquito blood meal and ensuring transmission. This remarkable ability of mature GIE to circulate through the spleen is due to the important deformability that they acquire during the transition between stages IV to V [9,11,12]. By contrast, immature GIE are particularly stiff, which likely contributes to their sequestration by mechanical retention [9]. Therefore, modulation of GIE mechanical properties plays a key role in their microcirculatory behaviour and it has been proposed that interfering with mature GIE filterability through spleen capillaries may represent a novel way to block parasite transmission [4,9]. However, mechanisms mediating the switch in GIE deformability late in the maturation process are still elusive. The disassembly of the microtubule subpellicular network subtending the trilaminar membrane structure in the transition from stage IV to stage V gametocytes probably contributes to this process [12–15]. The switch in deformability is also linked to the de-association of the parasite-derived STEVOR proteins from the infected erythrocyte membrane [9]. These processes must be tightly controlled and signalling likely plays a regulatory role. In uninfected erythrocytes, changes in phosphorylation status, including phosphorylation by cAMP-dependent kinase A (PKA), are known to regulate mechanical properties of the erythrocyte membrane [16,17]. For instance, phosphorylation of band 4. 1 by PKA may be central to the regulation of erythrocyte cytoskeletal organization and membrane mechanical properties [18]. PKA phosphorylation of dematin has also been shown to modulate the association between actin and spectrin in the erythrocyte cytoskeleton [19,20]. In infected erythrocytes, PKA activity results from both the human and the parasite enzymes [21]. During the parasite’s life cycle, plasmodial PKA activity is implicated in a wide variety of processes including P. berghei sporozoite motility and liver cell invasion [22], P. falciparum erythrocyte invasion by merozoites [23,24], or modulation of infected erythrocyte membrane permeability [25]. So far, there is no evidence for a regulatory role for cAMP-signalling during sexual development. However, adenylate cyclase alpha (PfACα) is highly expressed in gametocytes [26], and PKA activity is reportedly higher in gametocyte-producing parasites compared to parasites defective in gametocyte production [27], suggesting a potential role for cAMP-signalling in sexual development. Here, we have investigated the role of cAMP-signalling in modulating GIE mechanical properties. Using both genetic and pharmacological manipulation of cAMP signalling in conjunction with the microsphiltration method to assess the ability of GIE to circulate through inter-endothelial splenic slits, we show that a decrease in cAMP levels increases mature GIE deformability, and conversely, increasing cAMP levels increases GIE stiffness. These findings provide the proof of principle that molecules targeting phosphodiesterases (PDE) represent a novel drug class capable of blocking malaria transmission. To investigate whether PKA activity modulates GIE mechanical properties, we assessed the filterability of GIE using the microsphiltration method, which mimics the physical constraints experienced by infected erythrocytes in the splenic microcirculation [28,29]. In this system, increased retention rates correspond to decreased erythrocyte deformability and impaired filterability. We treated stage III GIE with KT5720 and H89, two independent and widely used PKA inhibitors that also block a few other kinases [30,31] and that have already been shown to inhibit PKA activity in P. falciparum [21,24,32]. Before incubation with these inhibitors, approximately 94% of stage III GIE and 30% of stage V GIE were retained on the microspheres (Fig 1A and S1 Fig), confirming the retention rates previously observed [9]. Importantly, incubation with H89 and KT5720 significantly decreased the retention rates of stage III GIE (P = 3. 10e-6 and 6. 10e-6 for H89 and KT5720, respectively; Fig 1A) consistent with PKA activity contributing to immature GIE stiffness, whereas incubation of stage V GIE with H89 did not alter their retention rates (S1 Fig). By contrast, stage III GIE retention rates were not affected upon incubation with compound 2, a highly selective inhibitor of apicomplexan cGMP-dependent protein kinase (PKG) [33], or with GGTI-298, an inhibitor of the cAMP-effector exchange protein activated by cAMP (EPAC) [34]. Furthermore, GIE filterability was not affected upon incubation with PKI-m, a membrane permeable inhibitor of the human PKA (PKA) that is a poor inhibitor of parasite PKA (PfPKA) [32]. This suggests that immature GIE filterability is modulated by PfPKA, and not by human erythrocyte PKA. To confirm this notion, we measured retention rates of a transgenic parasite that exhibits a down-regulation in PfPKA activity due to episomal overexpression of the regulatory (PfPKA-R) subunit of PfPKA (pHL-pfpka-r) [25]. cAMP binding to PKA-R liberates the catalytic subunit (PKA-C) from inactive R/C complexes and over-expression of PKA-R acts as a cAMP sink dampening complex dissociation, so decreasing PfPKA activity. Immunoblotting and immunostaining of gametocytes with specific antibodies indicated that PfPKA-R expression was significantly increased in pHL-pfpka-r transgenic parasites (Fig 1C, 1D and 1E). We note that two PfPKA-R specific bands are identified in gametocytes compared to a single band in schizonts [35], indicating that the R subunit undergoes translational modification during gametocytogenesis. Microsphiltration analysis of pHL-pfpka-r immature GIE showed a significant decrease in retention rates, similar to that observed upon incubation of wild-type GIE with H89 (P = 0, Fig 1B). To confirm that decreased retention rates were due to dampened cAMP-signalling, we measured deformability of pHL-pfpka-r immature GIE following incubation with the cell permeable, phosphodiesterase resistant cAMP analogue, 8Br-cAMP; raising cAMP levels restored retention rates to wild-type phenotype (Fig 1B). Loss of episomally derived PfPKA-R over-expression in pHL-pfpka-r transgenic parasites should result in a regain in PfPKA activity and restoration in the levels of retention associated with endogenous PfPKA expression. To promote shedding of the episome-encoded R subunit, the pHL-pfpka-r transgenic line was cultured for several generations without pyrimethamine selection required to retain the episome, leading to a new line called pHL-pfpka-r-wt. Immunoblotting and immunostaining of stage III GIE with specific anti-PfPKA-R antibodies confirmed that PfPKA-R expression in pHL-pfpka-r-wt had reverted to that of wild type levels, consistent with loss of the episome harboring the pfpka-r expression cassette (Fig 1C, 1D and 1E). Retention rates of pHL-pfpka-r-wt immature GIE were similar to wild-type GIE, indicating that the retention phenotype mediated by over-expressed PfR-induced down-regulation of PfPKA activity had reverted (Fig 1B). These results indicate that PfPKA-mediated phosphorylation contributes to immature GIE stiffness. To further demonstrate the contribution of PfPKA to immature GIE stiffness we probed membrane extracts of stage III and stage V GIE using a monoclonal antibody specific for canonical phospho-PKA sites (RRXS*/T*) (Fig 2A and 2B). The intensity of phosphorylation was 2-fold lower in stage V GIE compared to stage III, indicating that membrane components were less phosphorylated by PKA in mature than in immature gametocytes. At least five proteins displayed a higher degree of PKA site phosphorylation in stage III compared to stage V, suggesting that these PKA substrates are potentially involved in mediating the membrane rigidity phenotype (Fig 2A and 2B). Reduced PKA phosphorylation at stage V indicates a drop in cAMP levels accompanies the switch in deformability that occurs at the transition between immature and mature stages. The degree of cAMP-mediated phosphorylation can be increased by treatment with calyculin A, a serine/threonine phosphatase inhibitor (known to inhibit P. falciparum phosphatase-1-like activities [36,37]) that diminishes dephosphorylation of PKA substrates. Incubation of stage V GIE with calyculin A increased phosphorylation intensity at 2-fold to levels observed in stage III (Fig 2A and 2B). Consistently, incubation of stage V GIE with calyculin A markedly impaired their filterability by increasing the retention rates in microspheres up to 90% (P = 0. 0038), whereas it did not significantly affect filterability of uninfected erythrocytes (P = 0. 7; Fig 2C). To validate that increased retention rates triggered upon incubation with calyculin A corresponded to a decrease in GIE deformability, we visualized the shape of GIE as they flowed through the matrix by adding a paraformaldehyde-fixation step to the microsphiltration experiment (Fig 2D and 2E). 30% of untreated GIE maintained their original shape, whereas 70% displayed a twisted and deformed shape, likely reflecting their ability to squeeze and slide between microspheres [9]. Upon incubation with calyculin A the proportion of deformed GIE decreased from 70% to 40% (Fig 2D and 2E). To rule out any cytotoxic effect of calyculin A on gametocytes that could un-specifically result in higher cell rigidity, we measured parasite lactate dehydrogenase activity immediately and 72 h after calyculin A treatment and validated that gametocyte viability was not affected (Table 1). Manipulating the phosphorylation status using calyculin A clearly affects GIE mechanical properties and given the documented effect of calyculin A on PKA activity in other systems [37], it is consistent with PfPKA activity in mediating gametocyte deformability. The above results suggest that changes in cellular cAMP levels influence GIE deformability via PKA activation. We first measured cAMP concentrations in MACS-purified GIE at immature and mature stages. Intracellular levels of cAMP decrease approximately five-fold in stage V GIE compared to stage III GIE (P = 0. 008; Fig 3A), while PfPKA-R levels are unaltered between immature and mature stages (Fig 3B). Thus, increased deformability of stage V GIE is accompanied by reduced PKA activity due to a decrease in cAMP levels. This notion was underscored by increasing cAMP levels via addition of 8Br-cAMP and measuring the filterability of stage III compared to stage V gametocytes. Upon incubation with 8Br-cAMP, retention rates of stage III GIE were not modified, whereas those of stage V GIE were proportionally augmented with increasing concentrations of 8Br-cAMP. High concentrations of 8Br-cAMP did not affect filterability of uninfected erythrocytes, indicating that 8Br-cAMP specifically affects P. falciparum mature GIE (Fig 3C). Analysis of Giemsa stained smears of stage V GIE upstream and downstream of the microsphere matrix showed unaltered male: female ratios indicating that both male and female gametocytes use cAMP to regulate infected cell deformability. The drop in cAMP concentration in stage V GIE might be a result of either reduced cAMP synthesis by adenylate cyclases, or increased degradation by phosphodiesterases (PDEs). In P. falciparum, four genes encode PDEs: PfPDEα (PF3D7_1209500. 1), PfPDEβ (PF3D7_1321500. 1), PfPDEγ (PF3D7_1321600) and PfPDEδ (PF3D7_1470500) [38,39]. We performed real-time RT-PCR to quantify mRNA levels for all PDEs in asexual blood-stages as well as in immature and mature gametocytes. PfPDEα and PfPDEβ are mainly expressed in asexual blood-stages, PfPDEγ is minimally expressed in all blood-stages, whereas PfPDEδ is highly expressed in stage V gametocytes (Fig 4A). Importantly, mRNA levels of PfPDEδ increase approximately two- to three-fold in stage V compared to stage III gametocytes. These results are consistent with expression data available at PlasmoDB (http: //www. plasmodb. org/), where PfPDEδ is annotated as almost exclusively expressed in stage V gametocytes [40,41]. To determine whether PfPDEδ is involved in triggering the switch in deformability observed in stage V GIE, we analysed retention rates for mature GIE from transgenic parasites in which the PfPDEδ gene had been deleted [42]. We first measured cAMP concentration in MACS-purified stage III and stage V GIE and found loss of PfPDEδ activity led to a two-fold increase in cAMP levels in both immature and mature GIE (Fig 4B). Microsphiltration analysis of mature GIE from the PfPDEδ-mutant line showed a significant increase in retention rates (Fig 4C), indicating that this phosphodiesterase participates in regulating cAMP levels in mature gametocytes and hence GIE deformability. As an alternative way to investigate the effects of raising cAMP levels in GIE, we used pharmacological agents such as forskolin, an activator of mammalian adenylate cyclase, and zaprinast, an inhibitor of PDEs. Although zaprinast is well known as an inhibitor of mammalian PDE5, a cGMP phosphodiesterase, it is known to increase cAMP levels in human erythrocytes [43], and can inhibit both cAMP- and cGMP-PDE activities in P. falciparum (Table 2) [39]. To validate the effect of these molecules on intracellular levels of cAMP, we measured its concentration in MACS-purified stage V GIE and found a two-fold increase in cAMP levels in cells treated with either compound (P = 0. 029 and 0. 024 for forskolin and zaprinast, respectively; Fig 5A). Importantly, incubation of stage V GIE with either forskolin or zaprinast markedly increased microsphere retention rates by up to 82% (P = 0) and 86% (P = 0. 0002), respectively. Both reagents showed no significant effect on filterability of uninfected erythrocytes (P = 0. 49 and 0. 20 for forskolin and zaprinast, respectively; Fig 5B). Upon incubation with forskolin or zaprinast, the proportion of paraformaldehyde-fixed mature GIE that exhibit a deformed shape as they flowed through the matrix was decreased to 45% and 31%, respectively, compared to 70% of untreated cells (Fig 5C). The ensemble indicates that pharmacological agents that raise levels of cAMP affect mature GIE deformability impairing their ability to pass through an in vitro model for splenic filtration. PDEs have been well studied as potential drug targets in relation to numerous diseases. For example, the PDE5 and PDE6 inhibitor sildenafil (Viagra) is a widely used to treat erectile dysfunction. As a first step to address the potential of sildenafil to impair the circulation of P. falciparum mature gametocytes in humans and thereby block malaria transmission to mosquitoes, we measured the cAMP concentration in MACS-purified stage V GIE following incubation with sildenafil. We found that 100 μM sildenafil triggered an approximately four-fold increase of intracellular levels of cAMP, leading to levels similar to those of immature GIE (P = 0. 0068; Fig 6A). An increase in cGMP levels was also measured following sildenafil treatment (S2 Fig), consistent with our observations that both zaprinast and sildenafil can inhibit both cAMP and cGMP hydrolytic activities (Table 1). Upon incubation with different concentrations of sildenafil from 10 nM to 100 μM, retention rates of stage V GIE increased in a dose-dependent manner and reached 92% retention at 100 μM (Fig 6D). At 100 μM the proportion of paraformaldehyde-fixed GIE that exhibit a deformed shape as they flowed through the matrix decreased to 29%, compared to 70% of untreated cells (Fig 6B and 6C). Importantly, mature GIE exhibited greatly reduced filterability with more than 75% retention with 1 μM sildenafil (666. 7 ng/ml), which approximately corresponds to the reported peak serum concentration reached in humans after 60 min following 100 mg oral dose (Cmax 440 ng/ml) [44,45]. The filterability of uninfected erythrocytes was not significantly affected at these sildenafil concentrations indicating that it specifically affects infected erythrocytes (P = 0. 7012; Fig 6D). A novel paradigm that recently emerged postulates that the dynamics of immature P. falciparum GIE sequestration in the extravascular spaces of bone marrow and release of mature forms into peripheral circulation through bone marrow endothelial slits depends on an increase in GIE deformability at the transition from immature to mature stages [6,9]. The ability of mature P. falciparum GIE to circulate and pass through narrow blood capillaries and splenic slits depends on the deformability of both the parasites and the infected erythrocytes. Here, we have provided new mechanistic insight into the regulation of GIE deformability, and importantly, by demonstrating that GIE deformability is altered by zaprinast and sildenafil treatments we provide the proof of concept that PDE inhibitors could open new avenues towards the design of malaria transmission-blocking drugs. Stiffer immature gametocyte stages have high levels of cAMP, but become more deformable upon inhibition of PfPKA either pharmacologically or by overexpression of the PfPKA regulatory subunit. Deformability was not affected upon incubation with GGTI-298, making unlikely that cAMP regulates GIE stiffness via activation of PfEPAC [35]. So one mechanism by which raising cAMP levels induce GIE stiffness is via activation of PfPKA and its possible substrates could be parasite proteins involved in mediating the deformability of the parasite itself, a parasite protein (s) that remodels the erythrocyte membrane, or a protein (s) of erythrocyte origin. For instance, glideosome associated protein (GAP) 45 and myosin A, which are components of the microtubule subpellicular network subtending the trilaminar membrane structure in immature gametocytes, have been identified as PKA substrates in asexual stages [12,46]. In uninfected erythrocytes PKA-mediated phosphorylation of dematin and protein 4. 1 decreases their ability to promote spectrin binding to F-actin, therefore modulating mechanical properties of the erythrocyte [18,20]. In GIE these proteins may be phosphorylated by PfPKA in immature stages and may become dephosphorylated by parasite phosphatases in deformable stage V gametocytes. The effect on deformability of the phosphatase inhibitor calyculin A is consistent with dephosphorylation playing a role. The detection of five bands showing a decrease in PKA site phosphorylation in mature stages suggests that more than one PKA substrate likely contributes to regulation of GIE deformability. Besides proteins from the erythrocyte cytoskeleton, parasite-encoded STEVOR proteins are also attractive candidates to be PfPKA substrates. They have been shown to impact on the deformability of erythrocytes infected with sexual and asexual P. falciparum parasites, and the switch in deformability during gametocyte maturation is linked to a de-association of STEVOR proteins from the erythrocyte membrane in mature stages [9,47]. Interestingly, three serine residues and one threonine residue conserved across the entire STEVOR family are predicted to be PKA sites (cbs. dtu. dk/services/NetPhosK). Thus, phosphorylation of STEVOR proteins and its impact on STEVOR subcellular localisation and/or their interaction with erythrocyte cytoskeleton could be dependent on PKA-mediated phosphorylation and future studies will address this point. In addition, the mature parasite-infected erythrocyte surface antigen (MESA) is also expressed in sexual stages and interacts with the erythrocyte cytoskeleton, where it binds to protein 4. 1 [48,49]. The presence of four classical PKA phosphorylation sites in MESA suggests that this protein might also be a target of PKA-mediated changes in GIE mechanical properties. PfPKA-mediated phosphorylation of proteins associated with the erythrocyte cytoskeleton, or parasite proteins located in the erythrocyte membrane implies that the parasite kinase might be exported into the erythrocyte cytosol. PfPKA-R and PfPKA-C sequences do not have a recognizable PEXEL/HT motif and whether they are secreted remains hypothetical; however, there are proteins that lack a clear secretion signature that make up the non-PEXEL exportome [50]. Furthermore, in the absence of compelling secretion data for PfPKA-R and PfPKA-C, we entertain the possibility that PfPKA effects on erythrocyte membrane deformability may be mediated indirectly, through PfPKA-dependent phosphorylation of other, as yet to be identified, secreted parasite effectors. We found that the overall GIE cAMP concentration drops at the transition between immature and mature gametocyte stages, concomitant with the switch in deformability necessary for transmission. We established that raising cAMP levels in stage V GIE with forskolin, zaprinast or sildenafil rendered them stiff, like immature GIE. Zaprinast and sildenafil treatment of stage V GIE clearly led to a rise in cAMP levels and sildenafil also induced a small increase in the amount of cGMP (S2 Fig). Consistent with these observations, retention rates of stage V GIE proportionally augmented with increasing concentrations of 8Br-cGMP (S2 Fig). However, cGMP levels do not change between stage III and V gametocytes [42] and moreover, inhibition of the cGMP-dependent protein kinase (PfPKG) with the specific inhibitor compound 2 did not alter stage III GIE retention rates at a concentration 20-fold higher than required to inhibit erythrocyte invasion by merozoites [33]. Nonetheless, cGMP could impact indirectly on GIE deformability via crosstalk with cAMP signalling, as already reported for uninfected erythrocytes [43]. These observations validate the use of sildenafil to increase mature GIE stiffness. Several PDE inhibitors have been developed and used as therapeutic agents, and PDE5 has received considerable attention over the last 10 years, with three selective inhibitors now on the market (sildenafil, vardenafil, and tadalafil) [45]. In humans sildenafil acts by inhibiting both PDE5 and PDE6, and we found that in P. falciparum-infected erythrocytes this inhibitor clearly led to a rise in cAMP levels. Consistently, it increased GIE retention in an in vitro model for splenic filtration, demonstrating that administration of PDE inhibitors could be a new way to block parasite transmission to mosquitoes. Interestingly, previous studies with PfPDEδ-mutant line pointed to an essential role for cGMP-signalling in P. falciparum gametogenesis and ookinete formation in the mosquito vector [42,51]. This suggests that inhibition of plasmodial PDEs with sildenafil or derived analogues has the potential to raise both cAMP and cGMP levels resulting in a block in both gametocyte transmission via changes in GIE deformability and on ookinete development in mosquitoes. Our observations provide an opportunistic approach towards the discovery of new malaria transmission-blocking drugs, by taking advantage of the wealth of clinical data available for sildenafil, which been approved by the Food and Drugs Administration and is widely used in humans with little side effects to treat erectile dysfunction. In opposition to the Ehrlich’s “magic bullet” which consists of targeting pathways that are essential for parasites, but absent in humans, this strategy, referred to as “inverted silver bullet” [52], open new avenues towards the design of novel interventions to halt the spread of malaria to humans. The P. falciparum clonal lines B10 and 3D7 (clones of NF54), and the transgenic lines pHL-pfpka-r, and PfPDEδ- clone 4 have been described elsewhere [25,42,53]. Parasites were cultivated in vitro under standard conditions using RPMI 1640 medium supplemented with 10% heat-inactivated human serum and human erythrocytes at a 5% haematocrit [54]. Synchronous production of highly specific gametocytes stages was achieved according to described protocol [55]. For the isolation of gametocytes, culture medium was supplemented with 50mM (final concentration) N-acetylglucosamine (GlcNAc) from day 0 onwards and medium replacement was continued for 5 days to eliminate the asexual stages. Gametocyte preparations were enriched in different experiments by magnetic isolation using a MACS depletion column (Miltenyi Biotec) in conjunction with a magnetic separator. To measure cAMP and cGMP levels, 6. 106 GIE were purified by magnetic isolation and the cell pellets were incubated with drugs at 37°C, centrifuged at 1,500 x g for 5 min and washed with PBS. Sample diluent containing detergents to lyse the cells, inactivate endogenous phosphodiesterases and stabilize the cyclic nucleotides was added to the pellet for 10 min at room temperature, as described in the kits protocol (FluoProbes, powered by Interchim). To avoid eventual interference with the assay stemming from haemoglobin contained in the red blood cell lysate, proteins were precipitated with 5% trichloroacetic acid (TCA) for 10 min on ice, and the precipitate was removed by centrifugation at 1,500 x g for 10 min. The supernatant was carefully removed and transferred to a clean tube. TCA was removed by four successive extractions with water-saturated Diethyl ether. The cyclic nucleotides content was measured after acetylation using a commercially available cAMP High Sensitivity Chemiluminescent Assay Kit or cGMP High Sensitivity Chemiluminescent Assay Kit (FluoProbes, powered by Interchim), cyclic nucleotides content was expressed as picomoles of cAMP or cGMP per 4. 107 cells. Synchronized cultures containing 1 to 5% GIE were incubated 15 min to 2 h at 37°C with 10 nM to 150 μM 8Br-cAMP (8-Bromide-cyclic adenosine-monophosphate), 100 to 150 μM forskolin, 65 μM zaprinast, 50 nM Calyculin A, 10 nM to 100 μM sildenafil citrate, 10 μM compound 2 (4-[7-[ (dimethylamino) methyl]-2- (4-fluorphenyl) imidazo[1,2-a]pyridin-3-yl]pyrimidin-2-amine), 10 μM H89,10 μM KT5720,10 μM PKI-m (Protein Kinase Inhibitor myristoylated), 10 μM GGTI (GeranylGeranylTransferase I) 298 trifluoroacetate salt hydrate. None of the compounds reported above, except Compound 2, PKI-m and KT5720, affected stage V GIE viability measured as parasite lactate dehydrogenase (pLDH) levels [56]. Stage V GIE were treated with compounds at the highest dose, for the indicated times, washed to remove the compounds and assayed for pLDH activity both immediately and after 72 h incubation at 37°C. All reagents were purchased from Sigma-Aldrich or Euromedex, except Compound 2 that was provided by DB. Calibrated metal microspheres (96. 50% tin, 3. 00% silver, and 0. 50% copper; Industrie des Poudres Sphériques) with 2 different size distributions (5- to 15-μm-diameter and 15- to 25-μm-diameter) composed a matrix used to assay infected erythrocyte deformability under flow, as described [28,29]. Suspensions of synchronized cultures containing 1% to 5% GIEs were perfused through the microsphere matrix at a flow rate of 60 mL/h using an electric pump (Syramed _sp6000, Arcomed_ Ag), followed by a wash with 5 mL of complete medium. The upstream and downstream samples were collected and smeared onto glass slides for staining with Giemsa reagent, and parasitaemia was assayed by counting 2000 erythrocytes to determine parasite retention versus flow-through. Retention rates of uninfected erythrocytes were monitored after labelling a subpopulation of erythrocytes with PKH67 (Sigma-Aldrich) according to manufacturer’s instructions. The proportion of labeled erythrocytes in upstream and downstream samples was determined by fluorescence microscopy using a Leica DM 5000 B at 100X magnification. To visualize GIE shape during their flowing through the matrix, 1 mL of PBS/4% paraformaldehyde was added after perfusion of the GIE-containing culture on the microsphere matrix. After 5 min of incubation, fixed GIEs were separated from the microspheres by a 3-step decantation procedure, and GIE morphology was observed on a glass slide by light microscopy using a Leica DM 5000 B at 100X magnification. Microsphiltration experiments were performed in triplicate and 100 cells were counted per experiment. PDE activity in native parasite fractions was measured using a modification of a previously published method [57]. Briefly, parasites were frozen in liquid nitrogen and stored at -80°C until use. Parasites were resuspended in 500 μl lysis buffer (20 mM hepes and 250 mM sucrose, pH 7. 0), subjected to 5 cycles of freeze-thaw in liquid nitrogen and pelleted at 100,000 g for 30 min. Particulate fractions were resuspended in lysis buffer containing EDTA-free protease inhibitors (Roche). PDE assays were carried out in triplicate wells of a 96-well plate in the presence of [3H]-labelled cGMP or cAMP (GE Healthcare) for 30 min at 37°C. Reactions were terminated by boiling the plate for 1 min, followed by a 3 min centrifugation at 900 g. 1 unit of alkaline phosphatase was added to each well and incubated for 30 min at 37°C. [3H]-labelled guanosine was separated from the radiolabelled cAMP/cGMP substrate using ion exchange (BioRad AG 1 x 8 resin). Supernatants containing the [3H]-labelled guanosine product were added to scintillation fluid (Optiphase Supermix, Wallac). Scintillation was measured using a Wallac 1450 Microbeta Liquid Scintillation Counter (Perkin Elmer) and PDE activity was expressed in pmol cAMP or cGMP/min/mg protein. Inhibition assays were carried out in the presence of compounds dissolved in DMSO. PDE assays for specific activity and IC50 determination were carried out at a native lysate dilution that gave 30% cGMP/cAMP hydrolysis. RNA was isolated from purified GIE or asexual stages using Trizol (Invitrogen) according to the manufacturer’s instructions and treated with DNAse I (Roche). RNA was reverse-transcribed using Superscript II that was primed with random hexanucleotides (Invitrogen). Real-time PCR was performed using an ABI Prism 7900HT sequence detector (Applied Biosystems). Relative quantification of cDNA was performed using 2ΔCt method (User Bulletin 2, ABI, http: //www. appliedbiosystems. com). Triplicate PCR reactions were analyzed for each sample. Transmission-blocking antigen precursor Pfs48/45 (PF13_0247) and ookinete surface antigen precursor Pfs25 (PF10_0303) were used as markers of stage III and stage V gametocytes, respectively. Transcript abundance was compared using mean of ΔCt values calculated using ubiquitin-conjugating enzyme (PF08_0085) transcript (HK gene) as endogenous normalizer. Gene-specific primers used to profile the expression of PDEα, PDEβ, PDEγ and PDEδ were published in Wentzinger et al (2008) [39], and gene-specific primers used to profile the expression of Pfs48/45, Pfs25 and HK were published in Joice et al (2013) [6]. GIE were purified by magnetic isolation and pelleted by centrifugation at 1800rpm. To prepare membrane extracts, 1. 107 GIE were resuspended in 100 μl of PBS1X/1%Triton X-100, freezed at -80°C overnight and centrifugated at 16000 g for 5 min at 4°C. To prepare total extracts, 5. 106 GIE were used. Pellets were denatured in protein loading buffer 5 min at 100°C and were separated by 4–12% SDS-PAGE, transferred to PVDF membrane and blocked for 1 h in 5% nonfat dry milk. Immunoblots were probed overnight with a purified rabbit antiserum against PfPKA-R at 1: 16, a rabbit mAb anti-phospho-PKA substrate (RRXS*/T*, 100G7E, Cell Signaling) at 1/1000, a mouse mAb anti-HSP70 antibody at 1/1000, or a mouse mAb anti-Band3 antibody (Sigma) at 1/5000 followed by 1 hour with horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG secondary antibodies (Promega) at 1: 10 000. Detection step was performed using the Pierce chemoluminescence system (Pierce) following the manufacturer’s instructions. The levels of PfPKA-R or phospho-PKA were quantified by densitometry using the Quantity One analysis software (BioRad). For each sample, we then calculated the ratio of protein levels relative to loading control HSP70 or Band 3. GIE were air-dried on glass blood smears and methanol-fixed for 10 min at -20°C. After 1h pre-incubation in PBS1X/2% BSA, slides were incubated overnight with a purified rabbit antiserum against PfPKA-R at 1/50 and with AlexaFluor 594-conjugated goat anti-rabbit affinity-purified IgG (Molecular Probes) for 1 hour. Parasite nuclei were stained with Hoechst 33342 (diluted 1: 20000, Life technologies). Samples were observed at 100X magnification using a Leica DM 5000 B. Statistical significance for differences in cAMP concentration and in protein levels was established using student test and Wilcoxon Mann-Whitney rank sum test. Statistical significance for differences in retention rates was established using Wilcoxon Mann-Whitney rank sum test. Statistical significance for differences in proportion of GIE showing different shape was established using a Chi-square test. | Title: cAMP-Signalling Regulates Gametocyte-Infected Erythrocyte Deformability Required for Malaria Parasite Transmission Summary: Malaria transmission is ensured by deformable mature gametocyte-infected erythrocytes being taken up when a mosquito bites. Non-deformable immature gametocyte stages are sequestered in the bone marrow, as their lack of deformability would lead to their splenic clearance. In the present study, we apply nano-filtration technology to mimic splenic retention and demonstrate that deformability of transmissible mature stage V gametocytes is regulated by parasite cyclic AMP-dependent kinase signalling. Importantly, when we used drugs to raise cAMP levels we render transmissible mature gametocytes as stiff as non-transmissible gametocytes. In contrast, when we inhibit the cAMP-dependent kinase we render immature gametocytes more deformable. Thus, by two different approaches we confirm that the drop in cAMP levels in mature gametocytes leads to an increase in their deformability and hence more likely to circulate through the spleen. Our molecular observations have the potential to be translated into therapies for blocking malaria transmission by demonstrating that raising cAMP levels with sildenafil also known as "Viagra" renders mature gametocytes rigid. These findings provide the proof of principle that deformability of circulating gametocytes is targetable by pharmacological agents and as such, it provides a novel approach to prevent the spread of parasites. PDE inhibitors therefore represent novel drug leads potentially capable of blocking transmission and improving the worldwide fight to eliminate malaria from the human population. | 9,403 | 370 | lay_plos | en |
Summarize: CROSS-REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of application Ser. No. 08/111,988, filed Aug. 25, 1993, now U.S. Pat. No. 5,439,595. BACKGROUND OF THE INVENTION This invention relates to a water treatment method and an apparatus, and more particularly, to a method and apparatus for the removal from aqueous fluid of toxic and potentially hazardous organic compounds. The present method and apparatus exploit, synergistically, ultraviolet photolysis, the use of hydroxyl radicals, and microwave energy to, optimize the oxidation of organic contaminants in water. The use of ultraviolet light and oxidants, such as ozone and hydrogen peroxide, to produce hydroxyl radicals, is well-known. Such a technique has been used to enhance oxidation of organic contaminants in industrial waste water, groundwater and other aqueous solutions. Direct photolysis of organic compounds by intense ultraviolet light is also well known and is used extensively in the biocidal water treatment industry. Microwave radiation (electromagnetic waves having a wavelength between about 0.3 and 30 centimeters) is commonly used to induce rapid heating of materials from within by oscillatory stimulation of hydrogen and nitrogen atoms within water and organic molecules. Oxidation of organic contaminants by ultraviolet light or by chemical reaction with hydrogen peroxide ultimately yields innocuous products: carbon dioxide, elemental carbon, water and oxygen. It has now been found that exploitation of the above techniques in a single compact apparatus creates a potent oxidative water treatment method. Existing apparatus and methods, using ultraviolet (UV) light and reagents such as hydrogen peroxide to create hydroxyl radicals, are able to treat substantial volumes of water, on the order of hundreds of gallons per minute. In "first generation" systems of this sort, it was proposed that low pressure UV discharge lamps be encased in quartz tubes immersed in tanks of water to be treated. Hydrogen peroxide was added to the water, and the mixture was allowed to flow around the submerged lamps. Problems with rapid fouling of the lamps and low production of hydroxyl radicals in such devices soon became apparent. Second generation apparatus of the above type incorporated manual cleaning mechanisms, and the use of polymer coatings (such as "Teflon" PTFE) on the quartz sleeve, additional oxidizers (such as ozone), and catalyzing additives (such as TiO 2 ) to enhance the rate of radical production. Lasers also have been used in efforts to increase energy transfer efficiency. Some efforts were successful to some extent, but at the price of significantly greater complexity and cost. In known prior art apparatus and methods, the oxidizing reagent is added to the water prior to exposure of the mixture to the UV radiation. Since chemical oxidation is rapid, in such arrangements inorganic precipitates quickly form on the quartz sleeve or window surfaces, resulting in immediate and cumulative attenuation of the UV radiation. Moreover, in known prior art apparatus and methods, addition of the oxidizing reagent to the water prior to UV exposure results in dilution of the reagent, so that the photon density of the UV radiation reaching the oxidant molecules is reduced by preferential absorption by the water, scattering and absorption by entrained particles in the water, and absorption by solutes. Sluggish mixing of the solution during irradiation also minimizes contact of the few radicals that are produced close to the light source, resulting in a relatively inefficient and certainly less than optimal capability for contaminant destruction. The present invention provides a method and apparatus which addresses and obviates the above shortcomings of the prior art, by synergistically enhancing contaminant destruction by complementary techniques. SUMMARY OF THE INVENTION In one of its aspects, the present invention provides, for use in the treatment of waste water, an ionizing reactor for photochemically oxidizing organic compounds in aqueous solutions, using microwave-assisted photolysis and hydroxyl radical oxidation. The photolysis, production of hydroxyl radicals and the final hydroxylation reaction are all effected using a high pressure UV short arc lamp. Peroxide activation is made to take place in such a manner that a pure reagent is irradiated immediately prior to water contact, thus maximizing O 3 and HO 2 + and OH + radical production. The reagent is injected into a downstream hydroxylation chamber, where it is activated and mixed in a reaction zone with the pre-sensitized water, still under irradiation. The mixing is made to take place at a paraxial focus, where the ionization kinetics are most aggressive and the principal oxidative destruction of organic compounds occurs. The activated oxidizing reagent, containing free radicals, and the microwave and photosensitized water, are thus mixed vigorously at a point where incident concentrated deep UV radiation irradiates both fluids at once, to enhance the chemical degradation reaction. Thus, a principal feature of a preferred embodiment of the invention is the simultaneous activation of pure oxidizing reagent with direct UV light and secondary photolysis and sensitization of contaminated water with UV radiation emitted from the wall of a tube (preferably of quartz) and with microwave radiation from a microwave source (magnetron) positioned to direct high energy electromagnetic waves into the angular path of influent water circulating in a vortex around the tube. In its method aspect, the invention contemplates the use of a concentrated beam of deep spectrum ultraviolet light, which is manipulated using an optical array to simultaneously irradiate a reagent such as hydrogen peroxide and to irradiate out of fluid contact with the reagent (and preferably in an environment charged with microwave radiation) water to be treated. The water and the reagent are then mixed vigorously, under continued UV irradiation to optimize oxidation of water-borne contaminants. A prime advantage of the above-described peroxide photolysis ionizer is that a maximum number of free radicals are produced using one UV source, which is itself safely and efficiently located separate from the reaction chamber. Presensitization of the water by photolysis enhances the ultimate reaction of the contaminants with the free radicals during subsequent hydroxylation. Because the hydroxylation reaction occurs downstream from any light transmitting or reflecting surfaces, it does not contribute to precipitate fouling of those surfaces, a common problem in the prior art. As indicated above, all of the fluid to be treated is preferably made to converge at a narrow vertex where a concentrated beam of UV light of optimal wavelength is directed. This minimizes the energy needed to generate free radicals while maximizing the treatment volume capability of the apparatus. The present invention may be used to good advantage as part of a comprehensive water treatment system in an industrial setting requiring the removal and rapid destruction of recalcitrant contaminants from water, within a minimal space. In one of its embodiments, the present invention may be incorporated into a system comprising a coalescing separator for receiving waste water, a multi-stage turbo-aspiration unit, and one or more peroxide photolysis ionizers of the above-described type associated with the various stages of the turbo-aspiration unit. All of the above components, it has been found, can be mounted on the chassis of a small truck or trailer for portability. BRIEF DESCRIPTION OF THE DRAWINGS There are seen in the drawings forms of the invention which are presently preferred (and which constitute the best mode contemplated for carrying the invention into effect), but it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown. FIG. 1 is a side elevation view, in cross section, of a chamber assembly of the peroxide photolysis ionizer of the present invention; FIG. 2 is a front elevation view, in cross section, of an arc lamp and exemplary associated fiber optic transmission bundles and quartz tubes for use in the present invention; FIG. 3 is a top plan view of a ringjet water and reagent flow control flange used in the invention; FIG. 4 is a cross-sectional view of the ringjet water and reagent flow control flange shown in FIG. 3, taken along the line 4--4 in FIG. 3; FIG. 5 is a side elevation view of a photolysis chamber in accordance with the invention; FIG. 6 is a top view of a photolysis chamber in accordance with the invention; FIG. 7 is an end view, in cross-section, taken along the line 7--7 in FIG. 1; and FIG. 8 is a flow diagram of a water treatment system in accordance with the invention. DETAILED DESCRIPTION OF THE INVENTION Referring now to the drawings in detail, wherein like elements are designated by like reference numerals, there is seen in FIG. 1 an-ionizing reactor, designated generally by the reference numeral 10. The reactor 10 comprises, in general, a photolysis chamber assembly, designated generally by the reference numeral 12, and a hydroxyl reactor chamber, designated generally by the reference numeral 14. Referring now to FIGS. 5 and 6 in addition to FIG. 1, the photolysis chamber assembly 12 consists, in the illustrated embodiment, of a generally elongated cylindrical shell 16, preferably of stainless steel. Associated with the shell 16 is a water injection port 18. As is perhaps best seen in FIGS. 5 and 6, the port 18 is so arranged and oriented with respect to the longitudinal axis "A" of the shell 16 that a stream of water flowing through the port 18 will impinge on the curved interior wall 20 of the shell 16 and assume a helical or volute path, shown and designated diagrammatically in FIG. 5 as "V". Referring now to FIG. 6, it will be seen that in a transverse cross-sectional view, the port 18 enters tangentially with respect to the circular cross-section of the shell 16. Referring again to FIG. 5, it will be seen that the longitudinal axis "B" of the water port 18 is oblique with respect to the axis "A" of the shell 16, thus encouraging development of the desired helical or volute flow of water within the shell 16. Referring again to FIG. 1 and also to FIG. 6, the shell 16 is capped at one end by a curved influent endcap 22, which supports the water injection port 18 and provides tube ports 50, 52 and 54. Referring to FIG. 1, the other end of the shell 16 is capped with a conical effluent mixing endcap designated generally by the reference numeral 40, which is secured to the shell 16 by a camlock bezel 41. Access may be had to the hydroxyl chamber 14 and photolysis chamber 12 upon removal of the endcap 40. This can be accomplished, in the illustrated embodiment, by a 1/4 rotation twist of the camlock bezel 41, manually, using the handles 38 best seen in FIG. 7. A suitable "O" ring 42 is disposed between the endcap 40 and a camlock flange 43 disposed on the shell 16. Referring again to FIG. 1, associated with the shell 16 is a ringjet flange, designated generally by the reference numeral 24. The ringjet flange 24 is disposed within the shell 16 when the photolysis chamber assembly 12 is assembled, and includes a hyperbolic reflector body 32, with a quadratic surface, shaped to direct radiation within the shell 16 for maximum effect. The reflector body 32 is preferably fabricated of highly polished aluminum, sputter-coated with sapphire to provide abrasion and corrosion protection and to optimize spectral reflectivity. Bolts 34, seen in FIG. 4, secure the reflector 32 to the ringjet flange 24. Associated with the ringjet. flange 24 is a water jet control manifold 36 (seen in detail in FIG. 3 and 4 and described below). The ringjet flange 24 seats on a retainer flange 26 (FIG. 1), which is part of the shell 16, and serves to support the assembly made up of the ringjet flange 24 and the reflector body 32. The ringjet flange 24 and retainer flange 26 partition the photolysis chamber 12 from the hydroxylation chamber 14. A lip 44, projecting from the conical endcap 40 (FIG. 1) seats on the ringjet flange 24 to hold the flange in place when the conical endcap 40 is locked in place. Other aspects of the ionizing reactor 10 will now be described in detail. Referring now to FIG. 6, associated with the shell 16, at a position generally juxtaposed to the water port 18, is a magnetron 46. The magnetron 46 is associated with a microwave-transparent "window" 48 in the shell 16, which enables microwave radiation produced by the magnetron 46 to enter the shell 16 and impinge upon water entering the shell 16 through the port 18. A suitable cooling fan, not shown, or other suitable cooling arrangement, may be provided for the magnetron. Associated with the above-mentioned ports 50, 52 and 54 of the endcap 22 are quartz tubes 56, 58 and 60. Jam nuts 62, with suitable gasket features, secure the quartz tubes 56, 58 and 60 to the endcap 22 at the respective ports 50-54. The quartz tubes 56-60, it should be understood, are evacuated and sealed, and serve as optical light pipes. They extend directly from a light concentrating condenser 81 at the light source (to be described below) to the reactor 10. In the illustrated embodiment the quartz tube 56 traverses the length of the photolysis chamber 12 in a direction parallel to the axis "A," and passes through the hyperbolic reflector body 32 and ringjet flange 24 into the hydroxyl reactor chamber 14. Referring again to FIG. 1, the terminal ends of the quartz tubes 58 and 60 are sealed by quartz plano-concave lenses 63, and the terminal end of the tube 56 is sealed by a quartz plano-convex lens 57. The tubes are evacuated and made to retain vacuum to optimize transmission of UV light. The ports 52 and 54 and the quartz tubes 58 and 60, it should be understood, are positioned and arranged so that UV light conducted by the quartz tubes 58 and 60 irradiates water as it enters the photolysis chamber 12 along path "B" as shown in FIG. 6. Also, spurious UV light is diffused throughout the photolysis chamber 12 by Lambertian diffusion from the wall of the tube 56. Referring now to FIG. 2, an exemplary ultraviolet light source is seen. The illustrated source provides an arc lamp 64, disposed within an ellipsoidal reflector 66, both within a housing 68. A cross-slide mechanism 82, associated with the housing 68 and arc lamp 64, provides for focus and alignment adjustments for the arc lamp. Any suitable mechanism may be used for incremental adjustment of the position of the arc lamp 64. Suitable adjustment wheels or knobs 84 (for arc lamp alignment) and 86 (for focus) are provided. The arc lamp 64 may be a 350 to 1000 watt high pressure snort arc mercury-xenon lamp, of the kind presently commercially available from Advanced Radiation Corp., Ushio Corp. and Ultra Violet Products, among others. Referring again to FIG. 2, a quadfurcated fiber optic collector 88 is juxtaposed to the elliptical reflector 66 at a 90° angle from the beam splitting chopper 74. Approximately fifty percent (50%) of the UV light passes directly through the chopper 74 and is focused into the quartz tubes 56, 58 and 60 (or light conducting elements associated with them) by a condenser 90. The other approximately fifty percent (50%) of reflected UV light is collected and collimated by the fiber optic collector 88 and directed through separate fiber optic bundles 92 (four in the illustrated embodiment) to the hydroxyl reaction chamber 14. In the chamber 14, each of the bundles terminates in a sealed sapphire lens housing 94, best seen in FIG. 1. The lens housings 94 oppose each other at 90° angles, but their optical axes are preferably angled about 40 degrees (40°) with respect to the longitudinal axis "A" of the shell 16, to intersect at a paraxial focal zone. The application of UV radiation to the fiber optic bundles 92 and the quartz tubes 56, 58 and 60 in the above manner results in irradiation of the above-mentioned paraxial focal zone in the hydroxyl reaction chamber 14, and, by incident impingement and Lambertian diffusion, irradiation of the photolysis chamber 12. The photolysis chamber 12 is preferably also simultaneously subjected to microwave radiation produced by the magnetron 46 (as described above), so that water in the photolysis chamber 12 is both irradiated and sensitized by the concurrent microwave and UVphoton impingement. Referring now to FIGS. 1, 3 and 4, the manner in which activated reagent and sensitized water are directed to a paraxial focal zone in the hydroxyl reactor chamber 14 (where UV radiation also impinges on both fluids) will now be described. It is intended that the principal oxidation reaction produced by the reactor 10 occur at this locus, and oxidation may be further enhanced by high shear mixing in a downstream turbo-aspirated sparging apparatus, as will be described below. Referring now to FIGS. 1, 3 and 4, the ringjet flange 24 will be described in greater detail. Referring to FIGS. 3 and 4, the ringjet flange 24 is provided with and pierced by a circular array of arcuate water passages 96, 98, 100, 102 and 104, terminating in orifices 96'-104' (five in the illustrated embodiment). The passages 96-104 extend through the ringjet flange 24 at a preferred angle of about 20° with respect to the longitudinal axis "A" of the hydroxyl reactor chamber 14. The illustrated passages 96-104 and orifices 96'-104' are distributed around the periphery of the ringjet flange 24 at the same radial distance from its center. As is seen in FIG. 4, the respective longitudinal center lines "L" of the orifices 96'-104' converge at a locus (or focal zone), here designated "F". The oxidizing reagent, hydrogen peroxide, is introduced into the hydroxyl reactor chamber 14 through a passage 106 and a circular array of arcuate slit-like orifices 106' (such as, for example, weir plates), seen in dotted line in FIG. 3. Other specific configurations for the orifices 106' will-occur to those skilled in the art. The orifices 106' are preferably disposed concentrically within the circular array of the water passages 96-104, and are angled with respect to the axis "A" to direct reagent toward the focus, or focal zone, "F. " The reagent may be introduced to the reactor 10 under pressure, through ports such as the port 108 seen in FIG. 1, The port 108 is in communication with a passage 110 through the retainer flange 26. The passage 110 aligns and communicates with the passage 106, best seen in FIG. 3, and the passage 106 communicates with the orifices 106'. The orifices 106' are so positioned and arranged that reagent emerging from the orifices 106' does so at an angle with respect to the longitudinal axis "A" of the hydroxyl reactor chamber 14. Such a configuration produces the cone of reagent within or closely associated with the cone of water impinging at the same locus "F. " The UV light from the fiber optic bundles 92 and quartz tube 56 is also directed to the focal zone at the locus "F. " Thus, is will be seen that the oxidizing reagent (hydrogen peroxide) emerging from the orifices 106' into a zone adjacent the locus or focal zone "F" is activated by the incident UV light. The oxidizing reagent containing free radicals, and the microwaved and photosensitized water, are vigorously mixed, and incident concentrated radiation continues to irradiate both fluids as contact mixing takes place in the focal zone "F. " FIG. 8 illustrates a water treatment system in accordance with the invention, in which the above-described ionizing reactor 10 cooperates with a number of known components assembled in a unique manner, to provide efficient treatment of contaminated water. Referring now to FIG. 8, the system, designated generally by the reference numeral 112, includes a coalescing separator 114, into which influent is introduced at 116. Heavy sediments and immiscible fluids are mechanically separated from the influent and removed at the sediment drain 118. Lighter contaminants, such as floating hydrocarbons in the liquid phase, are drawn off at a conduit 120 to a collection and storage drum 122. The remaining water, still containing dissolved organic contaminants, is withdrawn from the separator 114 through the conduit 124, and pumped as input into a module containing a turbo-aspirated sparger 126. Off gas from the sparger is removed through a manifold 128, and the water output of the sparger is directed and pumped to the water injection port 18 of an ionizing reactor 10. Oxidizing reagent, such as hydrogen peroxide, is provided to the reactor 10 from a storage drum 130, by means of a metering pump 132 and conduit 134. The effluent from the ionizing reactor 10 is introduced into a second sparger 136, whose off gasses are drawn off into the manifold 128. The efflux from the sparger 136 is pumped through a conduit 138 to a third sparger 140 (also associated with the manifold 128), and from the third sparger 140 through a conduit 142 to a fourth sparger 144 (also associated with the manifold 128). Clean water is discharged from the fourth sparger 144 at a conduit 146. A turbo-aspirator 148 may advantageously be associated with each module containing a sparger 126, 136, 140 and 144. It will be appreciated that, although four spargers (and thus four sparging stages) are shown, the present invention may be used with other numbers of sparging stages. Various commercially available sparging units are suitable for use in the above-described system. The present invention may be embodied in other specific forms without departing from its spirit or essential attributes. Accordingly, reference should be made to the appended claims, rather than the foregoing specification, as indicating the scope of the invention. | Summary: Apparatus and a method are disclosed for decontaminating water, using an ionizing reactor. Water contaminated by organic compounds is introduced into a chamber in which it is concurrently irradiated by microwave and an ultraviolet source to activate it by photolysis. The water is then introduced to a hydroxyl reactor chamber. An oxidizing reagent, such as hydrogen peroxide, is irradiated by subjecting it to the UV source. The activated water and irradiated oxidizing reagent are then vectored to a locus at which they are mixed under continuing UV radiation from the source. The apparatus and method may be incorporated into a water treatment system employing existing contaminant extraction techniques, such as immiscible fluids separation and turbo-aspirated sparging. | 5,685 | 165 | big_patent | en |
Summarize: By. Sophie Jane Evans. Aged 13, she was given her first journal by her mother in the summer of 1832. Now, more than 180 years later, the early diaries of the future Queen Victoria are to go on show at Windsor Castle. In the books, the then-Princess speaks of the 'friendliness' of locals in the 'desolate, blasted and black' Midlands following the Industrial Revolution. First journal: Queen Victoria (pictured, right, as a teenager), was given her first journal (left) by her mother, the Duchess of Kent, in August 1832. It is among an array of royal treasures to go on show at Windsor Castle. She also comments on her school timetable, which saw her undertake lessons in history, geography, Latin and general knowledge. The diaries are among an array of royal treasures to go on display in an exhibition celebrating 100 years of the Royal Archives. Queen Victoria, who is Britain's longest serving monarch, was given her first journal by her mother, the Duchess of Kent, in August 1832 as they visited Powis Castle in Wales. The Duchess encouraged her daughter to record her impressions of the places they saw as they attended a series of educational tours across the country. Keeping a record: In her early diaries, the then-Princess speaks of the 'friendliness' of locals in the 'desolate, blasted and black' Midlands following the Industrial Revolution. Above, an extract from her first journal. Passionate writer: During her lifetime, the Queen even kept brief diaries in Hindustani - 13 of which survive in the Royal Archives today. She began to learn the language with the help of Mohammed Abdul Karim (pictured) During the tours, the Princess was met with an unfamiliar sight of the newly industrialised Midlands, prompting her to write: 'The men woemen (sic), children, country and houses are all black. 'But I can not by any description give an idea of its strange and extraordinary appearance. 'The country is very desolate every where; there are coals about, and the grass is quite blasted and black. I just now see an extraordinary building flaming with fire.' However, despite her bleak surroundings, the future Queen was apparently charmed by the people she met, writing, 'We have just changed horses at Wolverhampton a large and dirty town but we were received with great friendliness and pleasure.' Amazing: Queen Victoria was made Empress of India in 1877. Above, an extract from her Hindustani diary. In 1832, the Princess recorded her school timetable in her journals, revealing she would spend half an hour at 9 o'clock each morning writing in her diary before undertaking a variety of lessons. The monarch's early diaries mark the beginning of a passion for writing that would last a lifetime, seeing the production of more than 43,000 pages within 141 journals, as well as vast amount of personal and official correspondence. It was the Queen's legacy that in 1914 prompted the creation of a permanent home for all documents relating to the Royal Family and Royal Household. Dedicated: Britain's longest-serving monarch continued to keep a diary until her death in 1901. As well as recording her thoughts and observations of daily life in her personal journals, Queen Victoria corresponded frequently with members of her family in Britain and throughout Europe. She also communicated with ministers, ambassadors, heads of state and the Church. The monarch even kept brief diaries in Hindustani - 13 of which survive in the Royal Archives today. She began to learn the language ten years later after being made Empress of India in 1877. She was taught by one of her Indian servants, Abdul Karim, who later became her Indian Secretary and Munshi. Queen Victoria continued to keep a diary until her death death in 1901. Only 13 of the original volumes survive, dating from 1832 to 1836. Upon the Queen’s instructions, her daughter, Princess Beatrice, produced abridged copies of the remaining volumes, destroying the originals. In 1912, Queen Victoria’s grandson King George V decreed that ‘All the Royal Archives shall be kept...in the Round Tower’, and in 1914 the transfer of the records to their new home in the Round Tower at Windsor Castle began. The monarch's first journal is among more than 100 items featured in the book Treasures from the Royal Archives, which is being published by Royal Collection Trust on Saturday, May 17. It is also one of 25 documents being exhibited at Windsor Castle until January 25 next year. For more details, visit royalcollection.org.uk | Summary: Queen Victoria was given first journal by her mother in summer of 1832. It is among an array of royal treasures to go on show at Windsor Castle. In early diaries, the 13-year-old speaks of 'desolate, blasted' Midlands. But she was apparently charmed by the 'friendliness' of people she met. Monarch's diaries mark the beginning of her life-long passion for writing. | 1,083 | 100 | cnn_dailymail | en |
Write a title and summarize: Broadly-neutralizing monoclonal antibodies (bNAbs) may guide vaccine development for highly variable viruses including hepatitis C virus (HCV), since they target conserved viral epitopes that could serve as vaccine antigens. However, HCV resistance to bNAbs could reduce the efficacy of a vaccine. HC33. 4 and AR4A are two of the most potent anti-HCV human bNAbs characterized to date, binding to highly conserved epitopes near the amino- and carboxy-terminus of HCV envelope (E2) protein, respectively. Given their distinct epitopes, it was surprising that these bNAbs showed similar neutralization profiles across a panel of natural HCV isolates, suggesting that some viral polymorphisms may confer resistance to both bNAbs. To investigate this resistance, we developed a large, diverse panel of natural HCV envelope variants and a novel computational method to identify bNAb resistance polymorphisms in envelope proteins (E1 and E2). By measuring neutralization of a panel of HCV pseudoparticles by 10 μg/mL of each bNAb, we identified E1E2 variants with resistance to one or both bNAbs, despite 100% conservation of the AR4A binding epitope across the panel. We discovered polymorphisms outside of either binding epitope that modulate resistance to both bNAbs by altering E2 binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). This study is focused on a mode of neutralization escape not addressed by conventional analysis of epitope conservation, highlighting the contribution of extra-epitopic polymorphisms to bNAb resistance and presenting a novel mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. Hepatitis C virus (HCV) infects over 170 million people worldwide [1] and kills more people in the United States annually than HIV [2]. Appalachian regions of the United States saw a >350% increase in the number of new HCV infections from 2009–2012 [3] and recent outbreaks in the United States have been attributed to the rapid increase in injection drug use [4]. While direct-acting antiviral (DAA) therapy has revolutionized care for patients with HCV, control of the HCV pandemic remains challenging due to frequent reinfection in high-risk individuals who have achieved a sustained virologic response after DAA therapy [5], transmission of NS5A inhibitor-resistant HCV variants from individuals failing DAA therapy [6], and the high proportion (~50%) of infected individuals who are unaware asymptomatic carriers [7]. A major goal for the development of a prophylactic vaccine against HCV is stimulation of an immune response that is protective against a wide range of naturally occurring viral variants [8,9], which is a daunting challenge given the enormous genetic diversity of HCV [10–18]. Broadly neutralizing antibodies (bNAbs) are a useful guide for vaccine development, since they bind to relatively conserved viral epitopes, prevent successful entry of diverse HCV isolates, and have been associated with spontaneous clearance of HCV [19]. Despite the relative conservation of bNAb epitopes, polymorphisms conferring resistance to various bNAbs have been identified [20–24], and increasing evidence has shown that polymorphisms distant from bNAb binding sites can modulate E1E2 resistance [20,22,24]. BNAb resistance polymorphisms have been identified by various methods, including alanine-scanning mutagenesis, mapping of longitudinal sequence evolution in infected humans [22], and passage of replication competent virus (HCVcc) in vitro in the presence of bNAbs [21,23], but an efficient method to identify common naturally-occurring resistance polymorphisms in circulating E1E2 variants has not been available. Recently, we and others have observed significant variation in sensitivity of natural E1E2 variants to a diverse panel of monoclonal bNAbs and HCV-infected sera [24,25]. When we compared the rank orders of neutralization of a diverse array of 19 genotype 1 HCVpp by individual bNAbs, distinct relationships between antibodies were observed, allowing grouping of all bNAbs into three distinct clusters of functionally-related antibodies, and suggesting that common E1E2 determinants of neutralization sensitivity are shared between bNAbs within each cluster [24]. In that study, we identified E2 polymorphisms conferring resistance to most antibodies falling in a group we called neutralization cluster 1, which included bNAbs that are known to target the CD81-binding site of E2. We were previously unable to explain the functional relationship between antibodies in a second group that we called neutralization cluster 2. Surprisingly, this cluster includes the potent human bNAbs HC33. 4 and AR4A, although their described binding epitopes are at opposite termini of the E2 protein [26,27]. HC33. 4 is a human monoclonal antibody that binds to a continuous epitope near the N-terminus of E2, at amino acids (aa) 412–423, commonly known as ‘epitope I’ [28]. Recently, aa 408 was also shown to be a HC33. 4 binding residue [29]. AR4A, also a human monoclonal antibody, binds to a conformational epitope including the C-terminal, membrane proximal region of E2 as well as residues on E1. BNAbs like HC33. 4 targeting ‘epitope I’ have been shown to neutralize HCV by blocking E2 interaction with CD81 [30–35], while AR4A does not appear to interfere with this interaction [26]. Despite the clearly distinct binding epitopes of these two bNAbs, and possibly differing mechanisms of neutralization, we hypothesized that shared E1E2 resistance polymorphisms to these antibodies would explain the unexpected correlation between neutralization profiles of HC33. 4 and AR4A. No obvious polymorphisms mediating this effect were identified in a small panel of E1E2 variants with varying HC33. 4 and AR4A sensitivities, so we developed a larger panel of more than 100 E1E2 variants as well as a statistical approach to identify natural polymorphisms that were associated with resistance to each bNAb. Using these tools, we identified polymorphisms conferring resistance to HC33. 4 and AR4A individually, as well as polymorphisms outside of either binding epitope that confer resistance to both bNAbs by modulating binding to the HCV co-receptor, scavenger receptor B1 (SR-B1). To construct a library of E1E2 genes to predict relationships between amino acid sequence and neutralization sensitivity, we cloned more than 700 naturally occurring HCV genotype 1 E1E2 genes. Of these cloned E1E2s, 113 produced HCV pseudoparticles (HCVpp) that were functional in repeated tests when co-transfected with an HIV NL4. 3Δenv-Luc reporter genome, as previously described [24]. The resulting library includes 71 subtype 1a and 42 subtype 1b E1E2 variants isolated from a total of 27 unique donors. It is not known why the majority of cloned E1E2 variants were nonfunctional in HCVpp, but this has also been observed in other studies [36]. We analyzed genetic variation in our functional E1E2 panel to confirm that it is representative of circulating strains. Loci of greatest amino acid variation of the panel across E1E2 mirror those of a reference panel of 643 genotype 1 HCV isolates from GenBank, with the greatest amino acid diversity observed in hypervariable region 1 (HVR1) of E2 (Fig 1A). As many as 9 possible amino acids are represented at some E1E2 loci. Overall, this neutralization panel of functional E1E2 variants contains 97% of amino acid polymorphisms present at ≥5% frequency in the Genbank genotype 1 reference panel. We first assessed variation at the known binding epitopes of the two bNAbs across the HCVpp panel. The HC33. 4 epitope varied at position 408, while the AR4A epitope was 100% conserved across the panel at all known binding residues (Fig 1B). We then quantitated the fraction unaffected (Fu) of each HCVpp in the presence of each bNAb by measuring hepatoma cell entry of HCVpp in the presence of 10 μg/mL HC33. 4 or AR4A relative to entry in the presence of nonspecific human IgG (Fig 1C). Neutralization was assessed at a single concentration of bNAb rather than with serial bNAb dilutions in order to increase the throughput of the assay and to minimize the quantity of bNAb required. We have previously shown that Fu values measured by this method are reliably quantitative, as they correlate significantly with IC50 values calculated from neutralization curves of the same HCVpp/bNAb combinations as well as with IC50 values calculated from neutralization curves of replication competent virus (HCVcc) [24]. Another recent study also confirmed strong concordance between neutralization results obtained using HCVpp or HCVcc [36]. Each bNAb showed a more than 100-fold variation in neutralization across the HCVpp panel, which was surprising given the conservation of the bNAb binding epitopes. HC33. 4 and AR4A were associated with a median (min-max) Fu of 0. 22 (0. 003–1. 1) and 0. 17 (0. 01–1. 15), respectively. Using a cutoff of Fu<0. 5, which is roughly equivalent to an IC50 cutoff of 10 μg/mL, HC33. 4 and AR4A neutralized 88% and 85. 8% of the HCVpp panel, respectively. As we previously observed using a panel of 19 HCVpp [24], there was a significant positive correlation between the rank order of sensitivity of the 113 HCVpp in this panel to HC33. 4 and AR4A (r = 0. 44 p = 7e-7) (Fig 2A). We identified E1E2 variants with exquisite sensitivity to both bNAbs (Fu < 0. 02 with either bNAb) as well as variants with high level resistance to both bNAbs (Fu >1), with other E1E2 variants distributed between those extremes. This correlation between neutralization profiles of the two bNAbs was surprising, given that they do not share binding residues (Fig 1B). To further confirm that the two bNAbs bind to distinct epitopes, we performed binding competition assays between the two bNAbs. E1E2 protein-coated ELISA wells were pre-incubated with a high concentration of either HC33. 4 or AR4A (blocking bNAbs), followed by biotinylated HC33. 4 or AR4A at a concentration selected to give 50% of maximal binding (EC50), with binding of the biotinylated bNAb detected using streptavidin-horseradish peroxidase. A ratio of binding of each biotinylated bNAb in the presence of blocking bNAb divided by binding in the absence of blocking bNAb was calculated (Fig 2B). As expected, each bNAb competed for binding with itself, but HC33. 4 and AR4A showed minimal competition for E1E2 binding with each other, confirming that the bNAbs bind to distinct epitopes. In spite of their shared resistance pattern, HC33. 4 more potently neutralized subtype 1a than subtype 1b HCVpp (Fu median = 0. 17 for 1a vs. 0. 27 for 1b, p = 0. 002), while AR4A displayed minimal difference in subtype neutralization (Fig 3A). The significant positive correlation between HC33. 4 and AR4A neutralization profiles observed with the full HCVpp panel was also observed on analysis of the subtype 1a-only (r = 0. 39, p = 7e-4) and subtype 1b-only (r = 0. 69, p = 1e-6) subsets of the panel (S1 Fig) We used novel (SNAPR) and established (LASSO) methods to identify E1E2 sequence determinants of resistance to HC33. 4 and AR4A. An E1E2 amino acid alignment was generated including sequences of each of the 113 variants in the panel. For the SNAPR method, due to the higher degree of similarity among E1E2 variants originating from the same HCV-infected donor, and a variable number of E1E2 variants contributed by each donor, neutralization and sequence data from variants from underrepresented donors were randomly selected for replication until the number of data points in the analysis representing each individual donor was identical. For each amino acid position across E1E2, all HCVpp were divided into groups according to the amino acid occupying that position, and the amino acid associated with lowest median Fu (greatest neutralization sensitivity) for each bNAb was identified. For each bNAb, at each position, a nonparametric (Wilcoxon rank-sum) test was used to compare the neutralization values of all HCVpp with E1E2 carrying the amino acid associated with the lowest mean Fu with the neutralization values of all of the other variants, generating a “SNAPR value” for each position (Fig 3B). While the replication of some sequence and neutralization data reduces the effect of over-representation of some donors, the method also artificially inflates the sample size with data that are not independent, so calculated p-values are artificially low. Therefore, the p-values themselves cannot be used to determine whether variation in neutralization sensitivity at an individual position is statistically significant. Rather, these" SNAPR-values" provide a metric for comparison between effects at different polyprotein positions. Because of the potential for subtype differences to dominate findings, grouped genotype (S2 Fig) and subtype 1a only analyses were performed separately for further focused investigation. SNAPR-values spanned approximately 10 and 20 orders of magnitude for HC33. 4 and AR4A, respectively (Fig 3C). We also re-analyzed the neutralization and sequence data using a method that considers multiple residues in combination—LASSO, without the replication of data required for the SNAPR analysis [37]. Of the 20 most likely resistance polymorphism position predictions from SNAPR for HC33. 4 and AR4A (Table 1) 5 positions (for HC33. 4) and 5 positions (for AR4A) were also among the 20 and 18 most likely LASSO predictions, respectively. Of note, position 408 was predicted by SNAPR to modulate resistance to HC33. 4 but not AR4A, which supports sensitivity and specificity of the SNAPR analysis since lysine (K) 408 is a known binding residue for HC33. 4 but not AR4A, and it is the only known binding residue for either bNAb that varies significantly across the neutralization panel. Position 408 was not among the top 20 LASSO predictions for HC33. 4. Given the distinct binding epitopes of HC33. 4 and AR4A (Figs 1B and 3C), and the imperfect correlation between neutralization profiles of the bNAbs (Fig 2A), it is not surprising that many of the loci predicted to modulate sensitivity to HC33. 4 and AR4A are not shared. Interestingly, SNAPR predicted positions 242,403, and 438 as determinants of neutralization sensitivity for both HC33. 4 and AR4A. Positions 242 and 403 were also predicted by LASSO to be determinants of sensitivity for both bNAbs (Table 1). To further investigate the 8 most likely SNAPR predictions for HC33. 4 and AR4A, we compared Fu values of HCVpp grouped by the amino acid present at each position, without the replicated neutralization data included in the SNAPR analysis (Fig 4). In the analysis for HC33. 4, E1E2 variants with methionine (M) vs. valine (V) at position 242 showed significant differences in neutralization sensitivity (median Fu 0. 10 vs. 0. 25, p = 0. 02) (Fig 4A). For AR4A, M vs. V at position 242 were also associated with significant differences in neutralization sensitivity (median Fu 0. 12 vs. 0. 27, p = 0. 02) (Fig 4B). Variation at the 242 position resulted in the 13th and 8th most extreme LASSO coefficients of any position in E1E2 for HC33. 4 and AR4A, respectively (Table 1). Variants with leucine (L) vs. phenylalanine (F) at position 403 showed significant differences in neutralization sensitivity to HC33. 4 (median Fu 0. 042 vs. 0. 22, p = 1E-3). (Fig 4A). For AR4A, L vs. F at position 403 were also associated with significant differences in neutralization sensitivity (median Fu 0. 08 vs. 0. 20, p = 0. 01) (Fig 4B). Variation at the 403 position resulted in the most extreme LASSO coefficients of any position in E1E2 for HC33. 4 and AR4A (Table 1). For HC33. 4, E1E2 variants with leucine (L) vs. valine (V) at position 438 also showed significant differences in neutralization sensitivity (median Fu 0. 096 vs. 0. 39, p = 3E-3). (Fig 4A). For AR4A, L vs. V at position 438 were also associated with significant differences in neutralization sensitivity (median Fu 0. 11 vs. 0. 44, p = 0. 01) (Fig 4B). To test SNAPR predictions, putative resistance polymorphisms at the 8 positions with the lowest SNAPR-values for each bNAb were introduced individually by site directed mutagenesis into 2–3 distinct wild type (WT) E1E2 variants in which they were not naturally present. These WT variants were each from subtype 1a and differed from each other prior to mutagenesis by an average of 42 amino acids (7%). Mutated E1E2 and corresponding WT variants were used to produce HCVpp, which were tested for neutralization by HC33. 4 (Fig 5A) or AR4A (Fig 5B). To control for experimental variation between HCVpp neutralization experiments, neutralization of each mutated and WT HCVpp pair was tested in at least 2 independent experiments. Experiments were considered independent only if independently produced HCVpp preparations (transfections) were used and independent neutralization assays were performed. M242V, L403F and L438V were predicted to modulate resistance to both HC33. 4 and AR4A, so these mutations were tested for effect on each bNAb. Four of the 8 polymorphisms predicted by SNAPR to confer resistance to HC33. 4 showed statistically significant effects after introduction by site directed mutagenesis. Notably, mutation of lysine (K) 408 to methionine (M) led to an increase in resistance (WT mean Fu of 0. 09 vs. K408M mean Fu of 0. 72, p<0. 0001), which was expected since K408 was recently identified by alanine scanning as a binding residue for HC33. 4 [29]. Mutation of leucine (L) 403 to phenylalanine (F) also led to a significant increase in resistance to HC33. 4. Curiously, mutation of L438 to valine (V), which was predicted by SNAPR to confer resistance to HC33. 4, instead conferred significantly increased sensitivity to the bNAb (WT mean Fu 0. 22 vs. L438V mean Fu 0. 04, p = 0. 004). Four of the 8 polymorphisms predicted to confer resistance to AR4A also showed statistically significant effects after introduction by site directed mutagenesis. As with HC33. 4, L403F conferred significant resistance to AR4A neutralization (WT mean Fu 0. 08 vs. L403F mean Fu 0. 23, p = 0. 007), and L438V conferred increased AR4A sensitivity (WT mean Fu 0. 20 vs. L438V mean Fu 0. 06, p = 0. 03). Notably, mutation of serine (S) 686 to threonine (T) and valine (V) 720 to isoleucine (I) also conferred significant resistance to AR4A. Neither S686 nor V720 fall at a known binding residue for AR4A, but they are 6 and 22 amino acids from known AR4A binding residues, respectively. Though polymorphisms at 242 were also predicted by SNAPR and LASSO to be determinants of resistance for AR4A and HC33. 4 (Fig 3C; Table 1), mutagenesis at this position did not confer a significant change in sensitivity to either antibody. Taken together, these results confirm that L403F and L438V modulate sensitivity to neutralization by both HC33. 4 and AR4A. All resistance polymorphisms except V720I that had been validated by introduction into neutralization sensitive E1E2 variants were reverted in 2–5 distinct E1E2 variants where they were naturally present (Fig 6). Mutated E1E2 variants and corresponding WT variants were used to produce HCVpp, which were tested for neutralization by HC33. 4 (Fig 6A) or AR4A (Fig 6B). Three of four polymorphisms that showed an effect on HC33. 4 when introduced into neutralization sensitive E1E2 variants also showed a significant effect when reverted in E1E2 variants where they were already naturally present. Mutation of M408 to the known HC33. 4 binding residue, K, resulted in significantly increased sensitivity to HC33. 4 neutralization (Wild type mean Fu 0. 75 vs. M408K Fu 0. 10, p = 0. 001). Mutation of F403 to L also increased sensitivity to HC33. 4. Mutation of V438 to L also conferred a small but significant increase in HC33. 4 sensitivity (Wild type mean Fu 0. 43 vs. V438L Fu 0. 38, p = 0. 02). This was unexpected because in other E1E2 variants, the reciprocal mutation of L438 to V had also conferred increased neutralization sensitivity, but the magnitude of the effect of V438L was very small (mean Fu fold change of 0. 9) relative to the magnitude of the effect of L438V (mean Fu fold change of 0. 2) Two of three polymorphisms that showed an effect on AR4A when introduced into neutralization sensitive E1E2 variants also showed a significant effect when reverted in E1E2 variants where they were already naturally present. Most notably, mutation of F403 to L and mutation of V438 to L conferred increased sensitivity to AR4A, just as they had for HC33. 4. Taken together, these results show that mutation of L403 to F and mutation of F403 to L confer reciprocal neutralization resistance and sensitivity effects on both HC33. 4 and AR4A, while mutation of L438 to V in some E1E2 variants and V438 to L in others confers increased sensitivity to neutralization by both HC33. 4 and AR4A. To measure the magnitude of the effect of L403F and L438V mutations on neutralization sensitivity, we measured neutralization of wild type 1a154 (H77), 1a154_L438V, and 1a154_L403F HCVpp by serial dilutions of HC33. 4 and AR4A (Fig 7A). As expected, the 1a154_L438V HCVpp variant was most sensitive to neutralization. Wild type 1a154 HCVpp showed a 3-fold increase in IC50 relative to 1a154_L438V for both antibodies, and 1a154_L403F HCVpp showed a 24-fold increase in IC50 relative to 1a154_L438V HCVpp for HC33. 4 and a 90-fold increase in IC50 for AR4A. We also confirmed the resistance phenotypes of the mutations using replication competent cell culture virus (HCVcc) (Fig 7B). Wild type 1a154,1a154_L438V, or 1a154_L403F E1E2 genes were cloned into a J6/JFH-1 HCVcc genome lacking E1E2 [38], and replication competent virus was produced from each chimeric strain. HCVcc neutralization results mirrored those observed with HCVpp very closely, with 1a154_L438V most sensitive to neutralization by each bNAb, wild type 1a154 8-fold more resistant to HC33. 4 and 7-fold more resistant to AR4A, and 1a154_L403F 40-fold more resistant to HC33. 4 and 24-fold more resistant to AR4A. To understand whether these changes in neutralization sensitivity were mediated by changes in binding of the bNAbs to E1E2, we performed an ELISA to measure binding of serial dilutions of the bNAbs to 1a154_L438V, 1a154, and 1a154_L403F E1E2 proteins (Fig 7C). No significant difference in binding of either bNAb to the E1E2 variants was observed, suggesting that differences in bNAb binding to E1E2 are likely not the mechanism by which L403F and L438V modulate resistance to neutralization by HC33. 4 and AR4A. Using Chinese hamster ovary (CHO) cells stably expressing either human CD81 or human SR-B1 [33], we investigated relative binding of wild type 1a154 (H77), 1a154_L403F, and 1a154_L438V E2 proteins to these HCV receptors. We used previously described methods to clone these variants without E1 and with replacement of their transmembrane domain with a histidine tag, allowing their expression as soluble E2 (sE2) [39]. Serial dilutions of these soluble proteins were incubated with CD81-CHO, SR-B1-CHO, or wild type CHO cells, then labeled with anti-HIS and fluorescent secondary antibodies to allow detection of binding of sE2 on the cell surface. We were able to quantitate dose-dependent binding of sE2 to both CD81 and SR-B1 using this technique. Fig 8A shows flow cytometry histogram plots of binding of serial dilutions of 1a154 sE2 to CD81-CHO cells, relative to background binding to wild type CHO cells without CD81 or SR-B1. After normalizing for total sE2 protein input (S3 Fig), we compared binding of serial dilutions of 1a154,1a154_L403F, and 1a154_L438V sE2 proteins to SR-B1 and CD81 (Fig 8B). Remarkably, we saw a clear increase in binding of 1a154_L403F to SR-B1 relative to binding of wild type 1a154, and we saw a decrease in SR-B1 binding of 1a154_L438V, matching the hierarchy of neutralization resistance of these E2 variants. In comparing binding of the same variants to CD81, we observed a small decrease in binding of 1a154_L403F relative to 1a154, and a large decrease in binding of 1a154_L438V, confirming that the differences between 1a154 and 1a154_L403F binding to SR-B1 are not likely due to differences in protein input. We next compared binding of a matched, fixed concentration of 1a154 and 1a154_L403F sE2 to SR-B1 and CD81 in the presence of increasing concentrations of nonspecific IgG or HC33. 4 (Fig 8C). The 1a154_L438V sE2 variant did not have high enough baseline binding to allow accurate measurement of percent inhibition by HC33. 4, and we were also not able to study AR4A in this manner because it requires both E1 and E2 for binding. HC33. 4 reduced binding of 1a154 and 1a154_L403F variants to both SR-B1 and CD81 in a dose-dependent manner. It is not surprising that HC33. 4 inhibits both SR-B1 and CD81 binding, since a prior study of HC33. 4-like antibodies showed that some could block binding to both receptors [33]. The concentrations of HC33. 4 inhibiting 50% of binding to SR-B1 or CD81 (IC50 values) of 1a154 and 1a154_L403F sE2 were nearly identical, suggesting that the differing affinities of these proteins for SR-B1 and CD81 did not alter the percent binding inhibition of each by equivalent concentrations of mAb. Unlike HCVpp in neutralization assays, the sE2 variants are normalized for protein concentration, so it is also informative to consider absolute sE2 binding in the presence of mAb. To determine whether modulation of SR-B1 binding could mediate mAb neutralization resistance, we analyzed absolute SR-B1 binding of a fixed concentration of sE2 in the presence of varying concentrations of HC33. 4 (Fig 8D), and binding of varying concentrations of sE2 in the presence of a fixed concentration of HC33. 4 (Fig 8E). Comparison of sE2 binding of 1a154 and 1a154_L403F in the presence of increasing concentrations of HC33. 4 (Fig 8D), showed that 1a154_L403F sE2 bound more SR-B1 than an equivalent concentration of 1a154 sE2 at inhibitory but non-saturating concentrations of HC33. 4. We also measured binding of serial dilutions of 1a154,1a154_L403F, and 1a154_L438V sE2 proteins to SR-B1 after preincubation with a fixed concentration of HC33. 4 (40 μg/mL) (Fig 8E). As observed in the absence of antibody, multiple concentrations of 1a154_L403F sE2 incubated with a high concentration of HC33. 4 showed greater binding to SR-B1 relative to 1a154 sE2, and 1a154_L438V sE2 showed consistently less binding. Together, these results suggest that, in the presence of inhibitory but non-saturating concentrations of HC33. 4,1a154_L403F sE2 binds more SR-B1 than an equivalent concentration of 1a154 sE2, and 1a154_L438V sE2 binds less, providing a likely mechanism by which these polymorphisms could confer increased resistance or sensitivity, respectively, to mAbs whose mechanism of neutralization is interference with the E2-SR-B1 interaction. We have developed a high-throughput platform for measurement of neutralizing antibody breadth and prediction of HCV neutralizing antibody resistance polymorphisms. Despite the relative conservation of HC33. 4 and AR4A binding epitopes, with 100% conservation of known AR4A binding residues across the panel, we identified E1E2 variants with resistance to one or both bNAbs. We also identified amino acid polymorphisms in E2 conferring resistance to each bNAb individually, as well as polymorphisms outside of both binding epitopes that modulate resistance to both bNAbs. We determined that two of these polymorphisms, L403F and L438V, modulate resistance of both HCVpp and HCVcc to both HC33. 4 and AR4A. These mutations increase or reduce E2 binding to SR-B1, identifying a novel mechanism of broad bNAb resistance. It is interesting that HC33. 4 IC50 values calculated from inhibition of binding of 1a154 and 1a154_L403F to SR-B1 were nearly equivalent, despite the apparent differences in affinity of the two sE2 variants for SR-B1 (Fig 8C). This could be a limitation in the sensitivity of the binding assay, or alternatively could suggest that HC33. 4 binding affinity for sE2 is significantly higher than the affinity of even the 1a154_L403F-SR-B1 interaction. These binding inhibition IC50 values were higher than the neutralization IC50 values measured for the same variants, likely due to differences in the assays, such as the amount of E2-receptor interaction necessary to generate a detectable signal above background. We show that, despite the equivalent binding inhibition IC50 values of 1a154 and 1a154_L403F, differences in sE2 binding to SR-B1 are a likely mechanism of neutralization resistance, since the neutralization resistant variant, 1a154_L403F, binds more SR-B1 than the same amount of 1a154 sE2 in the presence of non-saturating concentrations of HC33. 4 (Fig 8D and 8E). As our ability to query larger sets of naturally occurring HCV isolates for their sensitivity to bNAbs increases, so does our understanding of determinants of bNAb resistance—a key barrier to developing an effective prophylactic vaccine against HCV. In a previous report, we found that bNAbs cluster into functional groups with respect to the HCV variants that they neutralize most and least potently. This clustering of bNAbs is determined at least in part by shared or overlapping binding epitopes, but we and others have shown that polymorphisms distant from known binding epitopes can also confer bNAb resistance [20,22,24]. This study provides evidence that these extra-epitopic polymorphisms play an important role in neutralization resistance of natural E1E2 isolates. Mutations arising within mAb binding epitopes tend to be an antibody-specific resistance mechanism, and cannot confer resistance to bNAbs with epitopes that are 100% conserved. Here we describe a novel mechanism that can confer resistance to multiple anti-HCV bNAbs, even if the bNAb binding epitopes are completely intact, by modulating E2 binding to SR-B1. To our knowledge, L403F and L438V are the first examples of a naturally-occurring mutations that confer resistance or sensitivity to bNAbs by this mechanism. L438 falls near the CD81 binding site of E2 [40], which is consistent with our finding that mutation at this site reduced sE2 binding to CD81, possibly also contributing to the increased bNAb sensitivity of L438V mutants. Introduction of L438V significantly decreased E1E2 fitness to mediate HCVpp or HCVcc entry into hepatoma cells (S5 Fig), which is consistent with the observed reduction in binding of 1a154_L438V sE2 to CD81 and SR-B1. We found that L403F increased binding to SR-B1 but decreased binding to CD81. Notably, despite these opposing binding effects, we observed a net increase in E1E2 resistance to neutralization by HC33. 4 and AR4A after introducing this mutation. The effect of L403F on SR-B1 binding may be dominant over the CD81-binding effect because the interaction of E2 with SR-B1 most likely occurs before binding of CD81 during HCV entry [41,42], or because of differences in relative expression of SR-B1 and CD81 on the surface of hepatocytes. This warrants further study, as it has potentially interesting implications for strategies to inhibit HCV entry with antibodies or small molecules. The binding epitope of HC33. 4 has been mapped in prior studies, and L403 and L438 were not found to be binding residues [28]. The binding epitope of AR4A is less clearly defined, but L403 and L438 were also not among probable AR4A binding residues [26]. Notably, L438 was identified as a contact residue for mAb AR3C in the crystal structure of AR3C/strain H77 E2 described by Kong et al [39], and another study showed that AR3C and AR4A do not compete for binding to E2 [26], Together, these data are all consistent with our finding that L403 and L438 are extra-epitopic for HC33. 4 and AR4A. Recent crystallization of the E2 protein core in complex with a bNAb has been informative [39]. However, large deletions in E2 to facilitate crystallization preclude analysis of many bNAb epitopes, including the HC33. 4 and AR4A epitopes. Given the difficulty and limitations of co-crystallizing HCV bNAbs with HCV E2, much of what we know about bNAb-E1E2 interactions will need to be inferred by a comprehensive approach including binding studies with alanine-scanning mutants as well as binding competition assays. This study shows the utility of an additional, complementary approach that can be used to measure neutralizing breadth, group functionally similar bNAbs, and identify bNAb resistance polymorphisms that may fall within or outside of known binding epitopes. These data are particularly relevant given studies in animal models suggesting that combinations of bNAbs may be necessary to provide sterilizing protection against HCV infection [43,44]. Based on their distinct binding epitopes, it would have been reasonable to assume that neutralizing breadth of bNAbs like HC33. 4 and AR4A would be greater if they were used in combination. That may still be true, but this study shows that an unexpectedly high proportion of HCV variants with resistance to one bNAb may also have resistance to the other, which could reduce the efficacy of this bNAb combination. While we were able to identify polymorphisms modulating resistance to multiple bNAbs, there were limitations to the study design and approach. We only sampled 97% of the naturally occurring polymorphisms that exist at a ≥5% threshold in a large set of Genbank HCV genotype 1 sequences. When the frequency threshold for polymorphism prevalence is reduced to ≥1%, the coverage is reduced to 78%. While SNAPR correctly predicted the 438 locus as a modulator of HC33. 4 and AR4A resistance, it incorrectly predicted that L438V would confer bNAb resistance, when in fact this mutation confers increased sensitivity to both bNAbs. The error may have arisen due to genetic linkage between the 438 locus and other resistance-determining loci in E1E2, since the LASSO algorithm, which adjusts for linkage, did not predict that the 438 locus is a determinant of neutralization sensitivity. Notably, the SNAPR algorithm accurately predicted position 408, a known binding residue, as a mediator of HC33. 4 resistance, while LASSO did not. Further testing would be necessary to more clearly determine whether SNAPR, LASSO, or a combination of the two methods would be best suited to predict resistance polymorphisms in HCV E1E2. While the effects of the L403F and L438V polymorphisms are significant, they are small in magnitude relative to the large variation in neutralization sensitivity observed between natural isolates, suggesting that combinations of polymorphisms likely play an important role in bNAb resistance. Even larger, more diverse E1E2 panels are required to reduce confounding from genetic linkage, to probe rarely occurring natural polymorphisms, and to better define the influence of combinations of polymorphisms on neutralization resistance. In conclusion, we have developed a large, diverse HCV neutralization panel and a statistical approach using amino acid sequence variation and neutralization sensitivity to identify bNAb resistance polymorphisms in E1E2. Despite conservation of HC33. 4 and AR4A binding epitopes across the E1E2 panel, we discovered variants with resistance to both bNAbs, identifying polymorphisms conferring resistance to each bNAb individually, as well as polymorphisms outside of either binding epitope that modulate resistance to both bNAbs. We determined that two of these polymorphisms, L403F and L438V, modulate resistance to HC33. 4 by increasing or decreasing E2 binding to SR-B1, which is a novel mechanism of bNAb resistance. This study highlights the important contribution of extra-epitopic polymorphisms to bNAb resistance, presenting a potential mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved epitopes. This diverse viral panel and novel computational pipeline are broadly applicable to future studies to define neutralizing antibody breadth, identify functionally-related bNAbs, and define mechanisms of bNAb resistance. HC33. 4 [29] was a gift of Steven Foung (Stanford University School of Medicine, Stanford, California. AR4A [27] was a gift from Mansun Law (The Scripps Research Institute, La Jolla, California, USA). Plasma samples obtained from HCV infected subjects in the BBAASH cohort [15,16,45], Irish Anti-D cohort [46], and Swan Project [47] were used to construct a library of genotype 1 E1E2-expressing lentiviral pseudoparticles using a high-throughput production and screening approach. The E1E2 region was PCR amplified from cDNA reverse transcribed from viral RNA purified from subject plasma and cloned into the expression vector pcDNA3. 2/V5/Dest (Invitrogen) using Gateway technology in a one-tube BP/LR reaction, as previously described [19]. HCVpp were produced by lipofectamine-mediated transfection of HCV E1E2 and pNL4-3. Luc. R-E- plasmids into HEK293T cells (ATCC) in 96-well plates as previously described [19]. Hep3B cells were exposed to transfected 293T supernatants in order to test for the presence of infectious HCVpp, as previously described [19]. HCVpp were considered infectious in the initial screen if infection of Hep3B cells (ATCC) in a 96 well format resulted in greater than 200,000 RLU of luciferase activity, which is >10X typical values obtained from infection with mock pseudoparticles. E1E2 variants included in the panel differed by at least one amino acid from every other clone contained in the library. Envelopes that displayed enhanced infection in the presence of neutralizing bNAbs (Fu >1. 2 with either bNAb) were not included in the analysis or in the description of library meta-data as these values most often resulted from HCVpp with poor infectivity. 18 of the 113 E1E2 variants in the final panel were previously described: 1a38,1a53,1a72,1a80,1a114,1a123,1a129,1a142,1a154,1a157,1b09,1b14,1b20,1b34,1b38,1b52 [19] and 1a116,1b21 [24]. Sequences of the remaining 95 E1E2 clones have been submitted to GenBank accession numbers KY565136—KY565230. Sanger sequencing of the entire length of the cloned E1E2 region was performed. Amino acid sequences from a nucleic acid MUSCLE alignment [48] were used to build a phylogenetic tree. Initial tree (s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with superior log likelihood value [49]. All trees are drawn to scale, with branch lengths measured in the number of substitutions per site, and all positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA6 [50]. Sequence logos were generated using VisSPAv1. 6 (http: //sray. med. som. jhmi. edu/SCRoftware/VisSPA/). Polymorphisms associated with bNAb resistance or sensitivity were introduced into at least two independent E1E2 clones. Mutants were created using the QuikChange Lightning Multi Site-Directed Mutagenesis Kit (Agilent) and Sanger sequencing was performed to verify that all mutants differed from parent clones at only the desired locus. For infectivity and neutralization testing of the panel of 113 HCVpp, 2,000 Hep3B cells per well were plated in 384-well white flat bottom tissue culture plates. For infectivity and neutralization testing of site-directed mutants 8,000 Hep3B cells per well were plated in flat bottom 96-well tissue culture plates and incubated overnight in a humidified CO2 incubator at 37°C. Media was removed from the cells the following day and replaced with 50μL of culture supernatant containing HCVpp (96-well plates) or 25μL of HCVpp supernatant (384 well plates). The plates were placed in a CO2 incubator at 37°C for 5 hours, after which the HCVpp were removed and replaced with 100μL of phenol-free Hep3B media (96-well plates) or 50μL of phenol-free Hep3B media (384-well plates) and incubated for 72 hours at 37°C. For 96-well plate infections, media was removed from the cells and 50 μL of 1x Cell Culture Lysis Reagent (Promega) added and left to incubate for >5 minutes then 45μL from each well were then transferred to a white, low-luminescence 96-well plate (Berthold) and read in a Berthold Luminometer (Berthold Technologies Centro LB960). Each sample was tested in duplicate. For 384-well plate infections, cells were lysed directly in the culture plate with 25μL of lysis buffer and luciferase activity measured using a BMG Labtech Fluostar Omega luminometer. A mock pseudoparticle (no envelope) was used as a negative control. The same procedure used to measure infectivity was employed, except that HCVpp were incubated with 10μg/mL of bNAb or serial dilutions of bNAb at 37°C for 1 hour prior to addition to the Hep3B target cells. Infections were performed in duplicate with the test antibody and nonspecific human IgG, the negative control. Murine Leukemia Virus (MLV) was used as a control for nonspecific neutralization. Fraction Unaffected (Fu) was calculated as RLU in the presence of test antibody/RLU in the presence of nonspecific human IgG. Each replicate RLUmAb value was divided by the average of two replicate RLUIgG values. % Neutralization was calculated as (1-Fu) x100%. To estimate the precision of Fu neutralization measurements, we compared Fu neutralization values of HCVpp measured in independent replicate experiments and observed a highly significant correlation (S6 Fig) Amino acid alignments were assembled as described in the phylogenetic analysis. To account for the uneven number of the infectious clones per human donor, neutralization values and corresponding E1E2 sequences were selected at random from each human donor and added to the initial data set until all donors were represented by an equal number of isolates. For each position in the alignment, HCVpp were grouped according to the amino acid encoded at that locus. The amino acid at each position associated with greatest bNAb sensitivity was identified by comparing median Fu values of the HCVpp in each amino acid group. Fu values for HCVpp in the most sensitive amino acid group were then compared to the Fu values of HCVpp with any other amino acid at the same position using a Wilcoxon rank-sum test. The resulting p-value is the SNAPR-value associated with that locus. Analysis was implemented using code developed for R, which is freely available upon request. The LASSO combines a prediction error term (the least squares error) with a model complexity penalty, which regularizes the model coefficients to perform variable selection and prevent over-fitting [37]. The two replicate fraction unaffected values were square root transformed, and the mean of these used as the outcome variable, which the model aims to explain using the amino acid sequence. The amino acids at each site were our explanatory variables, and these were encoded as indicator variables. Leave-one-out cross validation was used to select the optimal LASSO penalty (with the lowest mean-squared error), which gives the coefficients for each amino acid at each site, which were used as the LASSO results throughout. This was performed in R using the Lasso implementation from the package" glmnet" (https: //cran. r-project. org/web/packages/glmnet/). BNAb binding to E1E2 was quantitated using an enzyme-linked immuosorbent assay (ELISA) as previously described [51]. 293T cells were transfected with E1E2 expression constructs. 48 hours post-transfection cell lysates were harvested. Plates were coated with 500ng Galanthus nivalis (GNA) lectin (Sigma-Aldrich) and blocked with phosphate-buffered saline containing 0. 5% Tween 20,1% non-fat dry milk, and 1% goat serum. E1E2 cell lysates were added. BNAbs were assayed in duplicate 2. 5-fold serial dilutions, starting at 10 μg/ml. Binding was detected using HRP-conjuagated anti-human IgG secondary antibody (BD Pharmingen no. 555788). For binding competitions ELISA, E1E2 protein-coated ELISA wells were pre-incubated with 20 μg/ml of either HC33. 4 or AR4A (blocking bNAbs), followed by biotinylated HC33. 4 or AR4A at a concentration selected to give 50% of maximal binding (EC50), with binding of the biotinylated bNAb detected using streptavidin-horseradish peroxidase. A ratio of binding of each biotinylated bNAb in the presence of blocking bNAb divided by binding in the absence of blocking bNAb was calculated. HCVcc chimeras were generated as previously described [38,52]. Briefly, after digestion of the HCVcc backbone with AfeI (New England Biolabs), 1a154 (H77 strain), 1a154_L438V, and 1a154_L403F E1E2 genes amplified from library plasmids were inserted in frame using In-Fusion cloning (Clontech). 2μg of plasmid DNA was linearized using XbaI (New England Biolabs) then used for in vitro RNA transcription using the T7 MEGAscript kit (Ambion). RNA clean-up was performed using RNeasy mini kit (Qiagen), quantified using a NanoDrop 1000 spectrophotometer (Thermo Scientific), and stored at –80°C. 10μg of RNA was transfected into 1. 8e6 Huh7. 5. 1 cells (a gift of Charles Rice, The Rockefeller University, New York City, New York, USA) using Nucleofector Kit T (Amaxa) and plated in a 6-cm plate. Transfection supernatants were collected 4–11 days later and stored at -80°C. Supernatants were titered by serial dilution and infection of Huh7. 5. 1 cells. HCVcc neutralization assays were performed in triplicate as described elsewhere [38,52]. Briefly, human hepatoma Huh7. 5. 1 cells were maintained in DMEM supplemented with 10% fetal bovine serum and nonessential amino acids. 10,000 Huh7. 5. 1 cells per well were plated in flat bottom 96 well tissue culture plates and incubated overnight at 37°C. The following day, HCVcc were mixed with mAb (3-fold dilutions started at 50μg/mL) then incubated at 37°C for 1 hour. Media was removed from the cells and replaced with 50 μL of HCVcc/antibody mixture. The plates were placed in a CO2 incubator at 37°C overnight, after which the HCVcc were removed and replaced with 100μL of Huh7. 5. 1 media and incubated for 48 hours at 37°C. Medium was then removed and cells were fixed with 4% formaldehyde then stained for HCV NS5A using primary anti-NS5A antibody 9E10 (a gift of Charles Rice, The Rockefeller University, New York City, New York, USA) at 1: 2,000 dilution for 1 hour at room temperature. Cells were washed twice with PBS and stained using secondary antibody Alexa Daylight 488–conjugated goat anti-mouse IgG (Life Technologies) at 1: 500 dilution for 1 hour at room temperature. Cells were washed twice in PBS and then stored in 100μl PBS at 4°C. Images were acquired and spot forming units were counted for infection in the presence of mAb (HCVccSFUtest) or PBS alone (HCVccSFUcontrol) using an AID iSpot Reader Spectrum operating AID ELISpot Reader version 7. 0. Percent neutralization was calculated as 100% x [1- (HCVccSFUtest /HCVccSFUcontrol) ]. A truncated, soluble form of the 1a154 (H77) strain E2 ectodomain (sE2) that retains antigenticity and function as previously described [39], encompassing residues 384–645, was cloned into a mammalian expression vector (phCMV3_Ig Kappa_HIS, a gift of Leopold Kong, The Scripps Research Institute, La Jolla, California, USA) from plasmids containing H77 structural proteins. The vector allows expression of E2 protein with a C-terminal His tag as well as an N-terminal murine Ig Kappa leader signal for efficient protein secretion. H77 mutants, L403F and L438V, were created as described above and verified by Sanger sequencing. Each E2 construct was co-transfected with pAdvantage (Promega) into HEK293T cells and incubated for 72 hours at 37°C. Supernatant was collected at 48 and 72 hours, passed through a 0. 2μm filter, and concentrated using a regenerated cellulose centrifugal filter with a 10kDa cutoff (Amicon). Serial 6. 25 fold dilutions of each sE2 supernatant beginning with a 1 to 40 dilution were immobilized onto ELISA wells pre-coated with 500 ng Galanthus nivalis lectin (Sigma-Aldrich) and blocked with PBS containing 0. 5% Tween 20,1% nonfat dry milk, and 1% goat serum. Wells were probed with 0. 5 μg of a mouse monoclonal anti-6x His-tag antibody (ab18184, Abcam) and quantified using a HRP-conjugated goat anti-mouse IgG secondary antibody (ab97265, Abcam). The EC50 for each sE2 construct was calculated by nonlinear regression analysis and fold differences in EC50 used to normalize sE2 concentration in subsequent experiments. CHO-CD81 and CHO-SR-B1 binding experiments were carried out as previously described [33]. CHO cells expressing recombinant human CD81 or SR-B1 (a gift from Dr. Matthew Evans, Icahn School of Medicine, Mount Sinai, New York) were detached using PBS supplemented with 4mM EDTA and 10% FBS and washed in PBS containing 1% BSA. Cells (2E+05) were pelleted in a 96-well u-bottom plate and re-suspended in 2 fold serial dilutions of each sE2 construct (H77, L403F, and L438V). Following 30 minutes incubation on ice the cells were washed twice and incubated with 0. 5 ug of anti-6x His-tag antibody for another 20 minutes on ice. The cells were then washed again, re-suspended in an Alexa fluor 647-labeled goat anti-mouse IgG secondary antibody, and incubated on ice for 15 minutes. After a final wash, the cells were fixed with 1% paraformaldehyde and analyzed on a LSRII (Becton-Dickinson) using FloJo software (Tree Star). For mAb binding-inhibition experiments, sE2 was normalized for protein concentration, then diluted 1: 32 for SR-B1 binding, 1: 16 for CD81 binding. sE2 was preincubated with serial dilutions of HC33. 4 or nonspecific human IgG, then used to stain CHO cells as above. | Title: Extra-epitopic hepatitis C virus polymorphisms confer resistance to broadly neutralizing antibodies by modulating binding to scavenger receptor B1 Summary: Generation of an immune response that is protective against a wide variety of naturally occurring isolates is necessary for vaccines against highly variable viruses like hepatitis C virus (HCV). Two broadly neutralizing human monoclonal antibodies, HC33. 4 and AR4A, neutralize multiple highly divergent HCV isolates, raising hope that a vaccine against HCV is possible. Previous reports have defined the distinct, highly conserved sites on the viral envelope proteins where these antibodies bind. However, little is known about naturally occurring variation in sensitivity of different HCV isolates to these antibodies. We developed a high throughput assay and computational algorithm to evaluate over 100 naturally occurring HCV variants for their sensitivity to these two antibodies, identifying several resistance polymorphisms to each antibody which do not fall within their mapped binding sites. Furthermore, two of these polymorphisms modulate resistance to both antibodies by enhancing or reducing envelope protein binding to HCV co-receptor scavenger receptor B1 (SR-B1). By developing this broadly applicable platform, we have shown the important neutralization resistance conferred by changes distant from antibody binding sites, presenting a potential mechanism by which HCV might persist even in the face of an antibody response targeting multiple conserved sites. | 14,070 | 312 | lay_plos | en |
Write a title and summarize: Claude Guéant, le 11 mars 2011 à Paris. AFP/LUDOVIC Vous rêviez d'être ministre de l'intérieur. Revendiquez-vous d'être ministre de l'immigration? Claude Guéant : Je ne rêvais pas d'être ministre de l'intérieur. Cette idée ne m'était jamais venue à l'esprit avant le mois de novembre 2010, quand il en a été question une première fois. S'occuper à la fois de l'immigration et de l'intérieur est un gage d'efficacité. Il apparaîtrait paradoxal de dire que l'immigration est un problème majeur et de la confier à un secrétaire d'Etat. Marine Le Pen était sur l'île de Lampedusa, lundi 14 mars : vous-a-t-elle doublé? Je n'ai jamais eu l'intention d'y aller. Un déplacement à Lampedusa participe de l'instrumentalisation de l'immigration. A Vintimille en Italie, le 28 février, je suis allé vérifier l'efficacité du dispositif de contrôle à notre frontière et prendre des décisions pour le renforcer. C'est différent. Comment allez-vous "contrôler" les flux migratoires? Il est très important de maîtriser les flux migratoires. La France est un pays qui, traditionnellement, accueille les opprimés. Jamais le président de la République n'a dit qu'il était fermé à toute immigration. En revanche, ce gouvernement est hostile à l'immigration irrégulière. Mon travail est de lutter contre elle. Nous souhaitons bien accueillir les immigrés autorisés à venir chez nous mais nous ne voulons pas subir une immigration imposée. C'est la raison pour laquelle nous nous sommes toujours refusés à des régularisations massives, comme celles opérées entre 1997 et 2002 où un peu plus de 76 500 personnes avaient été régularisées. Depuis 2007, les régularisations se sont faites au cas par cas et à titre exceptionnel. Elles ont concerné 2800 personnes en 2008, et 2500 en 2009 et en 2010. En mettant l'immigration au premier plan, ne faites-vous pas le jeu du Front national? Le FN ne nous sert pas de boussole. Un gouvernement doit travailler en fonction de ce qu'il estime être nécessaire et il doit être à l'écoute de la population. Les Français ont le sentiment que les flux non-maîtrisés changent leur environnement. Ils ne sont pas xénophobes. Cela étant, ils veulent que la France reste la France. Ils veulent que leur mode de vie soit respecté, que la laïcité demeure à la base de notre pacte républicain. Ils veulent que l'intégration, pour les nouveaux venus, ou l'assimilation, pour les plus anciens, se fasse réellement, ils ne veulent pas de communautarisme. Les Français veulent que les nouveaux arrivés adoptent le mode de vie qui est le leur. | Title: "" "Les Français veulent que la France reste la France" " Summary: Pour le ministre de l¿intérieur et de l'immigration, qui a accordé au "Monde" son premier entretien depuis sa nomination place Beauvau, "certains fondamentaux" doivent être clarifiés sur la laïcité, comme la traçabilité de la viande hallalClaude Guéant, secrétaire général de l'Elysée depuis mai 2007, est ministre de l'intérieur depuis fin février, en remplacement de Brice Hortefeux. | 668 | 114 | mlsum_fr | fr |
Summarize: By. Suzannah Hills. The world's oldest crown believed to be more than 6000-years-old has gone on display for the first time in America. The ancient relic, which dates back to the Copper Age between 4000–3300 B.C., is shaped like a thick ring and features vultures and doors protruding from the top. It is believed the crown played a part in burial ceremonies for people of importance at the time. The crown was discovered in a remote cave in the Judaean Desert near the Dead Sea in 1961 among hundreds of other objects from the period. The world's oldest crown: This headpiece dating back to the Copper Age between 4000-3300 B.C. was found in a remote cave in the Judaean Desert in Israel in 1961. Moment of discovery: Pessah Bar-Adon from the Israel Department of Antiquities pictured in the so-called Cave of the Treasure in the Judaean Desert in Israel shortly after discovering the hoard of items from the Copper Age in 1961. Known as the 'Nahal Mishar Hoard', more than 400 objects were discovered by Pessah Bar-Adon and his fellow Israeli archaeologists in the cave which became known as the 'Cave of the Treasure'. The pieces included two clay statues of Gods - The Lady and Ram of Gilat - and a full array of Copper Age figurines made from copper, stone, elephant ivory and clay. There was also a scepter decorated with horned animals, clay goblets and bowls. It has been suggested that the hoard was the sacred treasure belonging to a shrine at Ein Gedi, some twelve kilometers away. The purpose of the hoard remains a mystery, it may have been to keep them protected in an emergency, although it is believed the objects may have been used in public ceremonies. Around 150 artefacts from the collection can be seen at New York University's Institute for the Study of the Ancient World as part of the 'Masters of Fire: Copper Age Art from Israel' exhibit which runs until June 8. Huge hoard: Archaeologists discovered more than 400 items hidden within the cave. Other discoveries: Among the other items discovered in the cave was a libation vessel in the shape of a woman carrying a churn, pictured left, and a fenestrated stand with three bowls and sculpted motifs, pictured right. Daniel M. Master, Professor of Archaeology at Wheaton College and a member of the curatorial team, said: 'The fascinating thing about this period is that a burst of innovation defined the technologies of the ancient world for thousands of years. 'People experimented with new ways to use not just copper, but also leather, ceramics, and textiles - sometimes successfully, sometimes not.' Jennifer Y. Chi, ISAW Exhibitions Director and Chief Curator, added: 'To the modern eye, it's stunning to see how these groups of people, already mastering so many new social systems and technologies, still had the ability to create objects of enduring artistic interest.' The exhibit also features objects from the Peqi’in Cave, another important discovery site. The most significant finds included are eight ossuaries, or burial containers, for human skeletal remains. Some were designed to look like human faces or figures and all are decorated with red stripes or zigzag patterns. These ossuaries held the bones of more than one person - the vast majority were men. Those who had their bones stored would have held positions of importance within society. For use in funerals: An ossuary with painted and sculpted facial features, pictured left, and a painted anthropomorphic ossuary with sculpted nose, stamped eyes, and gaping mouth, pictured right. Unusual: A libation vessel in the shape of a Ram carrying cornets | Summary: The crown was discovered in a cave in the Judaean Desert, Israel, in 1961. Archaeologists found more than 400 items hoarded in the cave. The ancient relics date back to the Copper Age - between 4000-3300 B.C. It is believed the items were used in burial ceremonies of important people. Around 150 items from the 'Nahal Mishar Hoard' are now on display. | 876 | 106 | cnn_dailymail | en |
Write a title and summarize: Neofunctionalization following gene duplication is thought to be one of the key drivers in generating evolutionary novelty. A gene duplication in a common ancestor of land plants produced two classes of KNOTTED-like TALE homeobox genes, class I (KNOX1) and class II (KNOX2). KNOX1 genes are linked to tissue proliferation and maintenance of meristematic potentials of flowering plant and moss sporophytes, and modulation of KNOX1 activity is implicated in contributing to leaf shape diversity of flowering plants. While KNOX2 function has been shown to repress the gametophytic (haploid) developmental program during moss sporophyte (diploid) development, little is known about KNOX2 function in flowering plants, hindering syntheses regarding the relationship between two classes of KNOX genes in the context of land plant evolution. Arabidopsis plants harboring loss-of-function KNOX2 alleles exhibit impaired differentiation of all aerial organs and have highly complex leaves, phenocopying gain-of-function KNOX1 alleles. Conversely, gain-of-function KNOX2 alleles in conjunction with a presumptive heterodimeric BELL TALE homeobox partner suppressed SAM activity in Arabidopsis and reduced leaf complexity in the Arabidopsis relative Cardamine hirsuta, reminiscent of loss-of-function KNOX1 alleles. Little evidence was found indicative of epistasis or mutual repression between KNOX1 and KNOX2 genes. KNOX proteins heterodimerize with BELL TALE homeobox proteins to form functional complexes, and contrary to earlier reports based on in vitro and heterologous expression, we find high selectivity between KNOX and BELL partners in vivo. Thus, KNOX2 genes confer opposing activities rather than redundant roles with KNOX1 genes, and together they act to direct the development of all above-ground organs of the Arabidopsis sporophyte. We infer that following the KNOX1/KNOX2 gene duplication in an ancestor of land plants, neofunctionalization led to evolution of antagonistic biochemical activity thereby facilitating the evolution of more complex sporophyte transcriptional networks, providing plasticity for the morphological evolution of land plant body plans. Gene duplication is thought to be one of the key drivers in generating evolutionary novelty. Following gene duplication, paralogs can undergo a process of neofunctionalization, supplying a genetic basis for morphological novelty [1,2, 3]. Transcription factors can undergo neofunctionalization via either a change in expression pattern or an alteration in functionality, e. g. the derivation of a repressor or inhibitor from an ancestral activator, or vice versa (e. g. [4]). Three amino acid loop extension (TALE) homeodomain transcriptional factors, characterized by having a homeodomain that has three extra amino acids between helices 1 and 2, are found in all eukaryotic lineages [5,6, 7]. Plant TALE homeobox genes are classified into two subfamilies, KNOTTED-like homeobox (KNOX) and BELL-like (BELL) [8]. Whilst Chlorophyte algal KNOX genes are of a single class, a gene duplication in a common ancestor of land plants, produced two classes of KNOX genes, class I (KNOX1) and class II (KNOX2) [8,9] (Fig. 1A). KNOX genes of flowering plants have been studied for over two decades, however, the functional consequences of the KNOX gene duplication have been largely unexplored. The first identified plant homeobox gene was Knotted1, a KNOX1 gene of maize [10]. Since, KNOX1 genes have been characterized in numerous flowering plants with a conspicuous loss-of-function phenotype being a failure in shoot apical meristem (SAM) maintenance. KNOX1 activity is also involved in maintenance of meristematic activity during leaf development, with prolonged activity in leaf margins observed in species with complex leaves and gain-of-function alleles result in more complex leaves. Thus, KNOX1 genes play a critical role in maintaining meristematic properties of cells in flowering plant sporophytes, the diploid generation of the land plant life cycle (reviewed in [11,12,13]). The KNOX1 genes of the moss Physcomitrella patens are only expressed in the sporophyte and mutants have decreased sporophyte growth, suggesting that KNOX1 genes have a conserved role in tissue proliferation during sporophyte development throughout land plants [12,13,14]. There is no evidence indicative of KNOX1 function in the gametophyte (haploid) generation in any characterized species, including the indeterminate meristems of the moss gametophyte, suggesting the role of KNOX1 is restricted to the diploid, sporophyte generation [14]. A functional distinction between KNOX1 and KNOX2 genes has been postulated from studies based on gene expression patterns in flowering plants. Northern blot analyses in maize demonstrated that KNOX1 gene expression is confined to less differentiated tissues whereas KNOX2 genes are broadly expressed in differentiating tissues and mature organs [9]. Similar broad expression profiles of KNOX2 genes have been reported in Arabidopsis [15,16] and tomato [17]. Characterization of spatial expression patterns in Arabidopsis revealed that KNOX2 genes have both overlapping and distinct expression patterns and that they are expressed in most tissues except for meristematic regions [15,18,19,20,21]. Despite several reports of expression patterns, comparatively little is known about KNOX2 gene function in flowering plants. One of the four Arabidopsis KNOX2 paralogs, KNAT7, is involved in secondary cell wall biosynthesis [18,19,21], and another, KNAT3, is reported to modulate ABA responses [22]. While these findings are consistent with the reported expression patterns, there exists a gap between broad expression patterns and known KNOX2 functions. For instance, unlike KNOX1 genes, which are important regulators of growth and development, it is not clear whether or not KNOX2 genes are involved in morphogenesis in flowering plants. These questions have gone unanswered owing to the paucity of functional studies on KNOX2 genes due to extensive genetic redundancy as noted by Truernit et al. [20]. From a wider perspective, a possible ancestral function of TALE homeodomain proteins is the regulation of diploid gene expression upon fusion of gametes, as is observed in the Chlorophyte alga Chlamydomonas reinhardtii and several fungi [23,24,25]. In C. reinhardtii the plus gamete expresses a BELL protein while the minus gamete expresses a KNOX protein; upon gamete fusion the KNOX and BELL proteins heterodimerize and regulate zygotic gene expression [25]. In the moss P. patens, KNOX2 genes are expressed in the egg cells and the sporophyte. Eliminating KNOX2 activity results in apospory, the development of a haploid body plan during the diploid generation, suggesting KNOX2 genes regulate the gametophyte-to-sporophyte morphological transition, a reflection of the hypothesized ancestral TALE homeodomain gene function [26]. Thus, both KNOX1 and KNOX2 mutant phenotypes in land plants are consistent with the hypothesis that ancestral function of KNOX genes was to regulate diploid gene expression. However, the seemingly different roles of KNOX1 and KNOX2 genes indicate functional diversification among land plant KNOX genes. To gain insight into developmental roles for KNOX2 genes in flowering plants and the genetic relationship between KNOX1 and KNOX2 classes, we undertook a genetic study of KNOX2 genes in Arabidopsis thaliana, a species in which KNOX1 gene function is well characterized. We discuss the implication of our findings on the impact of the gene duplication producing KNOX1 and KNOX2 paralogs in the course of land plant evolution. KNOX2 mutant phenotypes were characterized using null alleles (S5 Fig.). As reported previously [20], single mutants lack conspicuous aberrant phenotypes. Amongst double mutants, knat3 knat5 seedlings are distinguishable from wild type by a longer petiole and narrower lamina of cotyledons, and more deeply serrated leaf margins (Fig. 2A-B). Venation pattern is also affected in knat3 knat5 cotyledons (S6 Fig.). knat3 knat4 plants also have serrated leaves (Fig. 2I) and are sporophytically female sterile with abnormal integument development. While knat3 knat4/+ knat5 plants are also female sterile, knat3/+ knat4 knat5 plants are phenotypically wild type and produce viable seeds, facilitating characterization of segregating triple mutant plants. Selfed knat3/+ knat4 knat5 plants segregated small, dark-green plants with deeply lobed leaves (Fig. 2C-G and S14F Fig.), a phenotype reminiscent of gain-of-function KNOX1 alleles [12,13]. PCR-based genotyping indicated these plants were triple-mutant homozygotes (knat345). Since only a single mutant allele was available for each gene, we designed an artificial miRNA (amiRNA, [27]) targeting only KNAT4, amiR159-KNAT4 (S7A Fig.), and generated knat3 knat5 plants constitutively expressing amiR159-KNAT4 under the control of the Cauliflower Mosaic Virus 35S promoter (pro35S). pro35S: amiR159-KNAT4 knat3 knat5 lines closely resembled the identified knat345 plants (S8C-D Fig.). Another amiRNA, amiR159-KNAT345–1, was designed to target KNAT3, KNAT4, and KNAT5 (S7B Fig.). pro35S: amiR159-KNAT345–1 plants also show a deeply serrated leaf phenotype (S8E and S14H Figs.). We thus conclude that this is the triple mutant phenotype. Consistent with functional redundancy among these genes, dosage-dependent enhancement of the leaf serration phenotype was observed (Fig. 2H-L). Likewise, venation pattern in cotyledons is more severely affected in knat345 plants (S6 Fig.). Floral organs homologous with leaves are also affected. Sepals and petals are narrower and partially dissected in the knat345 mutant, and integument development is defective as seen in knat3 knat4 plants (Fig. 2M-N and S9 Fig.). Ectopic formation of tracheary elements is observed in knat345 embryo sacs (Fig. 2R). Although these genes are expressed in roots [15,16,20], the morphology of primary roots in knat345 plants appeared normal (S10 Fig.). A proKNAT5: KNAT5-GUS translational fusion line was generated to monitor expression patterns (Fig. 2S-W and S11 Fig.). In line with the mutant phenotypes, GUS activity was observed in developing leaves but excluded from the shoot apical meristem (SAM) (Fig. 2S-V and S11C Fig.). During early stages of leaf development, GUS activity was not detected in youngest leaf primordia but was observed in older leaf primordia (Fig. 2U). Reduced signal levels were observed in older leaves (S11B Fig.). Prolonged incubation detected GUS signal along cotyledon and leaf veins and in ovules (Fig. 2T and S11H-I Fig.). proKNAT5: KNAT5-GUS expression is nuclear in trichomes, supporting a role for KNAT5 in transcriptional regulation (S11 Fig.). A transcriptional fusion line, proKNAT4: GUS, was generated to examine KNAT4 expression patterns. Ten independent T1 plants were examined, all of which exhibited KNAT4 promoter activity in leaves but not in the SAM (Fig. 2X). A similar expression pattern has been described for KNAT3 using either a GUS reporter line or RNA in situ hybridization [15]. Exclusion of KNOX2 expression from the SAM is also supported by cell-type specific expression analyses of the inflorescence SAM (S3 Fig.). KNOX and BELL heterodimerization plays a pivotal role in regulating their activities as transcription factors [13]. We speculated that the lack of BELL partners may explain why no conspicuous phenotype has been described to date upon ectopic expression of KNOX2 genes [28] (S12 Fig.). The founding BELL gene, BELL1 (BEL1), and closely related paralogs, SAWTOOTH1 (SAW1) and SAW2, represent candidates for KNOX2 partners since loss-of-function phenotypes in ovules and leaf margins resemble those of KNOX2 mutants [29,30,31]. Physical interactions have been previously proposed between these BELL and KNOX2 proteins [22,29,32]. SAW1 and SAW2 are expressed in leaves but not in meristems [29] (S3 and S4 Figs.). Thus, we co-expressed SAW2 and KNAT3 throughout the SAM by trans-activating SAW2 under the control of SHOOT MERISTEMLESS (STM) regulatory sequences (proSTM>>SAW2; >> denotes the use of transactivation system hereafter) in pro35S: KNAT3 plants [33]. pro35S: KNAT3 proSTM>>SAW2 plants lack an embryonic SAM and resemble loss-of-function stm or stm knat6 mutant plants [34,35,36] (Fig. 3A-C, 3E). Combined expression of KNAT5 and SAW2 in the proSTM region resulted in a similar phenotype (Fig. 3D), confirming that the presence of both SAW2 and KNOX2 proteins simultaneously accounts for the phenotype. Collectively, these data indicate that concurrent expression, and by proxy, heterodimerization with BELL proteins, is important for KNOX2 function and that KNOX2 activity may thus be constrained by limited access to corresponding BELL partners. A mutation in KNAT3 suppresses the gain-of-function phenotype caused by ectopic expression of another BELL gene, BLH1, suggesting that BLH1 is likely a functional partner for KNOX2 proteins [37]. This prompted us to examine genetic interactions between BLH1 and BEL1-related BELL genes, and we found that bel1 blh1 double mutants show color changes in unfertilized gynoecia as seen in knat3 knat4 and knat345 plants (Fig. 3G-J and S9 Fig.). Thus, BEL1 and BLH1 play a redundant role in gynoecium development and perhaps act in association with KNOX2 genes. More comprehensive genetic analyses as well as expression analyses are required to assign specific roles to functionally redundant BELL genes. To further dissect BELL-KNOX interactions, plants expressing BELL and/or KNOX genes in the proSTM region were characterized. Among BELL proteins, PENNYWISE (PNY) and POUND-FOOLISH (PNF) are expressed in the SAM and act in conjunction with KNOX1 proteins to promote SAM activity [12,13]. As expected, plants expressing KNOX1 genes (STM or KNAT2) or PNY in the STM domain appeared wild type (S13B-D Fig.). In contrast, proSTM>>SAW2 plants displayed abnormal floral morphologies, such as fused sepals, reduced petals, and misshapen fruits (S13E, H Fig.), phenotypes often associated with reduced KNOX1 activity, e. g. weak stm mutants [38]. Flower development was not impacted in proSTM>>KNAT5 plants, but fused sepals are also observed in strong pro35S: KNAT3 lines (S12F Fig.). Concomitant expression of KNOX2 with PNY or KNOX1 with SAW2 did not enhance the KNOX2 or SAW2 overexpression phenotypes. We therefore conclude KNOX2 shows selectivity for BELL proteins in vivo. Loss-of-function and gain-of-function KNOX2 phenotypes are reminiscent of gain-of-function and loss-of-function KNOX1 phenotypes, respectively [12,13]. To characterize the relationship between the two gene classes, loss-of-function alleles for KNOX1 and KNOX2 were combined. Plants constitutively expressing an amiRNA targeting KNAT3, KNAT4, and KNAT5, pro35S: amiR159-KNAT345–1, in KNOX1 loss-of-function (stm or bp knat2 knat6) backgrounds were examined. Neither the meristem failure of stm mutants nor the KNOX2 loss-of-function mutant leaf phenotype was suppressed in these plants (Fig. 3F and Fig. 4A-B). Similarly, neither knat2 knat3 knat5 knat6 nor bp knat345 showed significant suppression of the KNOX2 loss-of-function mutant leaf phenotype and the bp inflorescence phenotype (Fig. 4F-J). Thus, loss-of-function phenotypes of KNOX1 and KNOX2 mutants are not due to ectopic activation of KNOX2 and KNOX1, respectively. Furthermore, BP, STM, and KNAT2 expression was not altered in knat3 knat5 plants (Fig. 4K-P), arguing against mutual repression between KNOX1 and KNOX2 genes. Deeply lobed leaves, a phenotype characteristic of gain-of-function KNOX1 alleles, occur in Arabidopsis plants where STM is driven by the leaf specific promoter, proBLS, proBLS: STM ([39]; S14B, C Fig.). These were crossed with loss-of-function KNOX2 plants (pro35S: amiR159-KNAT345–1) to generate plants with ectopic KNOX1 and reduced KNOX2 activities in the leaves. Compared to the parental lines, F1 plants harboring both transgenes displayed more extreme leaf margin elaboration (Fig. 4C-E). The additive effects, rather than epistatic interactions, suggest it is unlikely that the two subclasses negatively regulate one another. An attractive hypothesis for the antagonism between KNOX1 and KNOX2 is that they regulate shared downstream events in an opposite manner. The complex leaf of gain-of-function KNOX1 alelles is suppressed by reduction in CUP SHAPED COTYLEDON (CUC) transcription factor activity [40] (S14C Fig.). Two CUC genes are targeted by the miR164 family of miRNAs, and expression of miR164b in young leaves using regulatory sequences of the FILAMENTOUS FLOWER (FIL) gene (designated as proFIL), proFIL: miR164b, flattens the leaf margin in wild-type plants (S14D-E Fig.). Thus, the miRNA-mediated CUC regulation plays a key role in leaf margin elaboration [41]. Introduction of proFIL: miR164b also suppressed the leaf dissection phenotype in a knat345 mutant background (S14F-G Fig.). Among miR164 targets, CUC2 plays a major role in leaf serration development [41]. We find leaf serration is largely suppressed in the cuc2 knat345 and pro35S: amiR159-KNAT345–1 cuc2 backgrounds (Fig. 5A-C and S14H-I Fig.). In addition, constitutive expression of KNOX2 (pro35S: KNAT3) can partially suppress the proBLS: STM leaf phenotype (Fig. 5D-F). Thus, a common developmental program mediates both gain-of-function KNOX1 and loss-of-function KNOX2 leaf phenotypes. As observed in proBLS: STM plants, elevated levels of KNOX1 activity are often associated with increased leaf complexity (reviewed in [11]). In Cardamine hirsuta, a close relative of Arabidopsis, dissected leaf development requires KNOX1 expression in leaves, and additional KNOX1 expression leads to ectopic leaflet initiation [42]. We investigated the outcome of reduction in the level of KNOX2 activity in this species. In Cardamine, leaf shape exhibits heteroblasty with leaflet number increasing in later produced leaves. Although leaflet number can vary for a particular leaf position, the first and second leaves always consist of a single, undivided, lamina, and the third leaf typically consisting of three leaflets (S15A Fig.). An amiRNA, amiR159-KNAT345–2, was designed to target three Cardamine genes homologous to Arabidopsis KNAT3, KNAT4, and KNAT5. Constitutive amiR159-KNAT345–2 expression (pro35S: amiR159-KNAT345–2; S7C Fig.) results in plants with an extra lateral leaflet on the second leaf, observed in approximately 15% of individuals (27 of 188 plants derived from 6 independent lines), indicating KNOX2 activity influences complexity of dissected leaves in Cardamine (Fig. 5G-H). Furthermore, gain-of-function KNOX2 alleles (pro35S: KNAT3) simplify leaf shape, a phenotype particularly obvious in third leaves, which are undivided in strong lines (Fig. 5I-J and S15B Fig.). Thus, reduction or increase in KNOX2 activity leads to increase or decrease in leaf complexity, respectively, in Cardamine (Fig. 5 and S15 Fig.). This observation and the deduced KNOX1/KNOX2 antagonism are in consistent with the results in Arabidopsis. Expression of KNOX1 genes in leaves is correlated with increased leaf complexity and has been hypothesized to be influential in the evolution of leaf shape [42,43,44]. Given that seed plants leaves evolved from ancestral shoot systems, the ancestral seed plant leaf was likely complex, but fossil evidence and phylognetic analyses suggest that the ancestral angiosperm leaf may have been simple [45]. Regardless of the ancestral state, transitions from simple to more complex and vice versa have occurred repeatedly during angiosperm evolution [43,44,46]. In angiosperms, increase in leaf complexity is associated with increased KNOX1 activity while loss of KNOX1 activity in leaves results in decreasing complexity. While KNOX1 activity has been shown to play a pivotal role, other loci, such as REDUCED COMPLEXITY (RCO) in Cardamine and LEAFY (LFY) orthologues in legumes either contribute directly to modifying leaf shape or influence sensitivity to KNOX1 activity [11,47,48]. The lability of angiosperm leaf architecture may reflect that addition or loss of enhancer modules directing KNOX1 activity in leaves does not affect general plant viability. The present study demonstrates that KNOX2 activities can also influence leaf shape—leaf dissection increases with decreasing KNOX2 activity (Fig. 2) in a dose dependent manner—raising the possibility of whether changes in KNOX2 activity could also have contributed to the evolution of leaf morphology. Just as KNOX1 gain-of-function alleles result in increases in leaf complexity, novel gain-of-function KNOX2 alleles that alter temporal or spatial expression patterns within developing leaves could contribute to the evolution from complex towards simple leaf morphology, as suggested by our experimental results in Cardamine, via acquisition of leaf specific enhancers. Alleles resulting in loss of KNOX2 activity could also contribute to increases in leaf complexity as suggested by the dose dependent changes to leaf shape in Arabidopsis, however, this may be less likely due to pleiotropic effects of loss-of-function KNOX2 alleles. Intriguingly, in monilophytes KNOX1 gene expression is broadly similar to that of seed plants, with expression limited to less differentiated tissues including the shoot apical meristem, developing leaves, and procambial tissues [43,49,50]. KNOX2 gene expression has not been studied in detail, but similar to the situation in angiosperms, is reported to be throughout the sporophyte body [50]. In parallel with seed plants, simple leaves have evolved from more complex ancestral leaves within monilophytes [51]. Whether changes in KNOX1 or KNOX2 gene expression may be related to evolution of leaf form in monilophytes is presently unknown. One plausible explanation for the opposing action of KNOX1 and KNOX2 genes is an epistatic relationship between the gene classes. While non-overlapping expression patterns have been observed between KNOX1 and KNOX2 genes, we found no evidence for mutual repression. Alternatively, KNOX1 and KNOX2 proteins may interfere one another’s activity. Such a mode of action was proposed for KNATM in Arabidopsis and PETROSELINUM (PTS) /TKD1 in tomato, both of which are KNOX-related proteins that lack a DNA-binding homeodomain [52,53]. It is suggested that these mini KNOX proteins act as passive repressors and interfere with formation of a functional complex composed of canonical KNOX and BELL proteins. That KNOX2 function depends on the availability of appropriate BELL partners to be active, argues against a similar mechanism for the KNOX1/KNOX2 antagonism. Instead, our data favor a model whereby the antagonistic roles of KNOX1 and KNOX2 are at the level of opposing modes of transcriptional regulation. Since addition of a repressor domain causes a dominant negative phenotype, KNOX1 proteins can act as activators [39,54]. Conversely a KNOX2 protein, KNAT7, can repress transcription in a transient protoplast system [18,19], and a motif similar to known repression domains is found in the ELK domain of all KNOX2 proteins [55] (S16 Fig.). Comparison of KNOX1 and KNOX2 homeodomains reveals that the third helices, an important determinant of DNA binding specificity, are highly conserved, indicating similar DNA binding properties, at least in vitro (S16 Fig.). Concurrently expressed KNOX1 and KNOX2 proteins could thus conceivably compete with each other at some target genes. Indeed, a putative KNOX2-SAW2 complex can overcome endogenous KNOX1 activities in the meristem, as does a dominant-negative form of KNOX1 (e. g., TKN2-SRDX [39] and en298-STM [54]). However, as KNOX1 proteins have also been reported to act to repress gene expression, the activity of KNOX proteins may be modified by either BELL partners, or third parties, such as OVATE proteins that interact with KNOX/BELL heterodimers and influence both their cellular localization and transcriptional activity [32,37,56]. In a related scenario, KNOX1 and KNOX2 could act on different sets of paralogs of downstream targets. These hypotheses are not mutually exclusive, and depending on the cellular contexts, different modes of action could operate, as is the case for the yeast TALE protein, Matα2, which has different partners in different cell types (reviewed in [23]). Phylogenetic analyses indicate land plant KNOX1 and KNOX2 genes are derived from a single, ancestral KNOX gene. We hypothesize that subsequent to the KNOX1/KNOX2 gene duplication, accumulating structural differences endowed a new mode of action to at least one paralog. Therefore a possible evolutionary scenario could have an ancestral KNOX protein acting primarily as a transcriptional activator, with the evolution of a transcriptional repressor following gene duplication and neofunctionalization. The evolution of a repressor from an ancestral activator may be a common event, with several instances documented in plant transcription factor families [52,53,57,58,59,60]. Thus, within the context of land plant KNOX genes two types of negative regulators, in which the modes of repressor action are mechanistically different, may have evolved. Mini KNOX proteins act to inhibit KNOX activity by interacting with and sequestering BELL proteins [52,53], as opposed to antagonistic action at the level of downstream gene expression as we propose for KNOX2. The latter provides more flexibility due to the potential to act independently. Accompanying divergence in protein functionality, our data provides additional evidence for nearly complementary expression patterns of KNOX1 and KNOX2 genes in Arabidopsis thaliana. In contrast, in P. patens KNOX1 and KNOX2 genes exhibit both overlapping and distinctive expression patterns [14,26]. Changes in cis-regulatory sequences must have contributed to the establishment of complementary expression patterns during land plant evolution. Flexibility in gene regulatory networks governing meristematic maintenance and differentition engendered by the combination of changes in protein functionality and expression pattern could provide plasticity enabling morphological evolution. Heterodimerization between BELL and KNOX proteins is important for translocation of the complex into the nucleus [13]. BELL-KNOX2 heterodimerization may also be critical for providing specificity or increasing affinity of DNA binding (e. g. [61]). Although studies based on the yeast two-hybrid technique suggest physical interactions between BELL and KNOX proteins in a rather nonspecific manner [29,32], our genetic data suggest KNOX2 proteins interact in planta with a subset of BELL proteins, including those of the BEL1/SAW1/SAW2 clade. KNOX1 proteins rely on a distinct set of BELL proteins, e. g. PNY and PNF (reviewed in [12,13]). Due to an obligate heterodimerization requirement, the activity of a KNOX/BELL pair may be limited by the protein with the more restricted expression domain. In Arabidopsis KNOX2 functions appear to be regulated by restricted availability of corresponding BELL partners [29] (Fig. 3). Similar to KNOX genes, land plant BELL genes evolved from a single gene in the algal ancestor [9]. However, the diversification of paralogs followed a different trajectory in the two families since BELL genes do not fall into discrete functional clades (S17 Fig.). For instance, KNOX1-interacting BELL genes (PNY and PNF) form a sister clade with KNOX2-interacting BELL genes (BEL1 and SAW1/2). Moreover, genetic interactions implicate BLH1, from a phylogenetically distinct clade, as a KNOX2 partner since knat3 alleles suppress the phenotype induced by ectopic BLH1 embryo sac expression [37]. These phylogenetic relationships might be expected if the genome of the land plant common ancestor encoded a single BELL protein that interacted with both KNOX1 and KNOX2 proteins. As the BELL gene family diversified, subfunctionalization would have restricted interactions of BELL paralogs to specific KNOX1 or KNOX2 partners. The defining feature of land plants is the formation of an embryo—a multicellular diploid generation. One prominent feature within land plant evolution is the transition from a gametophyte-dominant life cycle to a sporophyte-dominant life cycle [62,63]. This process is regarded as progressive sterilization and elaboration of vegetative organs [62], and in flowering plants, the gametophyte is reduced to a ephemeral structure of only a few cells that is dependent on a sporophyte body that can live up to thousands of years. If the ancestral KNOX-BELL genetic program regulated gene expression in a single celled zygote [25], it follows that during the course of land plant evolution, the KNOX/BELL module has been recruited to control numerous aspects of sporophyte development, with KNOX1/BELL modules promoting meristematic maintenance and continued growth and KNOX2/BELL modules promoting differentiation. In some cases, there is resemblance to a presumed ancestral function, such as in P. patens where KNOX2 genes regulate the gametophyte-to-sporophyte morphological transition [14,26]. In other cases, however, KNOX/BELL modules direct the development of novel structures, such as sporophyte shoot meristems and leaves (Fig. 6), that evolved later in land plant evolution, suggesting the duplication and diversification of the KNOX/BELL genetic module is linked with the evolution of morphological diversity in the land plant sporophyte. Neofunctionalization, exemplified by opposing activities between KNOX1 and KNOX2 genes in Arabidopsis, may underlie the molecular mechanism of key innovations and modification of body plans in the land plant history, through elaboration of transcriptional networks. The role of TALE genes in fungi and Chlamydomonas can be viewed as promotion of cellular specialization in the diploid zygote and progression towards a meiotic state. The life cycle of land plants arose by an interpolation of mitotic divisions between fertilization and meiosis. Thus there is cell proliferation and a delay in meiosis in the diploid generation. KNOX1 genes prevent differentiation and maintain an undifferentiated state of the cells, enabling the cells to proliferate and develop a multicellular body in the sporophyte generation. In organisms with two heteromorphic multicellular generations, such as land plants, the developmental programs for each must be tightly controlled—a role suggested for KNOX2 genes in preventing the haploid gametophyte genetic program to be active during the diploid sporophyte generation in Physcomitrella. We hypothesize the duplication and diversification of the KNOX/BELL genetic module was instrumental in the evolution of a diploid embryo such that multicellular bodies develop in both haploid gametophyte and diploid sporophyte generations known as alternations of generations [25,26]. Alternations of generations have evolved independently in phylogenetically diverse eukaryotic lineages [64,65], prompting the question of whether similar TALE class genetic diversification may be found in these lineages. Arabidopsis thaliana accessions Columbia and Landsberg erecta (Ler) were used as wild type in most experiments. proKNAT2: GUS was generated in the C24 background and introgressed into Ler. Cardamine hirsuta ‘Oxford strain’ is a kind gift of A. Hay and M. Tsiantis. Plants were grown under long-day (18 hours light) or short-day (10 hours light) conditions at 20°C. knat3 and knat5 alleles are gift from V. Sundaresan and G. Pagnussat. bp-9 knat2–5 knat6–1 seeds are gift from V. Pautot. T-DNA insertion alleles for BELL and KNOX genes were obtained from the Arabidopsis Biological Resource Center (ABRC) or the Nottingham Arabidopsis Stock Center (NASC). Mutant and transgenic lines have been described previously: bp-9 knat2–5 knat6–1 [66]; stm-11 [67]; proBP: GUS [68]; proKNAT2: GUS [69]; Op: KNAT2 and Op: STM [39]; and proSTM: LhG4 [70]. The mutant and transgenic lines used in this study are listed in S1 Table. Homozygous mutant lines were identified by polymerase chain reaction (PCR) -based genotyping. Sequences of genotyping primers are available in S2 Table. The details of the transactivation system was previously described [33]. Multiple mutants combining knat3, knat4, and knat5 alleles were generated by crossing, and genotypes were confirmed by PCR-based genotyping. To generate bel1 blh1 double mutant, blh1 plants were crossed with bel1 plants, and the resulting F2 plants were examined. bel1 plants were identified based on self-sterility, and among them, plants with yellow gynoecia segregated and were confirmed to be bel1 blh1 double mutant plants by PCR-based genotyping. knat2 knat3 knat5 knat6 and bp knat345 plants were identified among F2 plants originating from a cross between bp knat2 knat6 and knat345 plants, and their genotypes were confirmed by PCR-based genotyping. cuc2 knat345 plants were identified in a F2 population derived from a cross between cuc2 and knat345 plants. To generate proFIL: miR164b lines in the knat345 mutant, self-fertile knat3 knat5 plants were transformed with the proFIL: miR164b construct, and tranformants were selected by resistance to herbicide Basta. Single insertion lines were selected and crossed with knat3/+ knat4 knat5 plants. Among F1 plants, self-fertile knat3/+ knat4/+ knat5 plants carrying the proFIL: miR164b transgene were selected, and F2 seeds were collected; proFIL: miR164b knat345 plants were identified in the resultant F2 population. To characterize the effects of the pro35S: amiR159-KNAT345–1 transgene in mutant backgrounds, the mutant plants were directly transformed with the pro35S: amiR159-KNAT345–1 construct, and transformants were selected by resistance to Basta. As stm null alleles are seedling lethal, heterozygous plants were used for transformation. More than twenty T1 plants for each background were examined, and phenotypes consistently observed among independent lines were reported. RNA was extracted, using the RNeasy Plant Mini Kit (Qiagen), from 10-day-old seedlings grown on half-strength MS medium supplemented with 0. 5% sucrose. RNA samples were treated with on-column DNaseI (Qiagen) and purified. SMARTScribe reverse transcriptase was used for cDNA synthesis (Clontech), and PCR reactions were performed using Ex Taq (Takara). Oligo sequences used for PCR reactions are described in S3 Table. amiRNAs were designed using the Arabidopsis pre-miR159a backbone (S7 Fig.) and synthesized (GenScript). For construction of the proKNAT5: KNAT5-GUS reporter construct, the genomic sequence spanning the KNAT5 locus (from the next upstream annotated gene [At4g32030] to the next downstream annotated gene [At4g32050]) was used, and the stop codon was replaced with the GUS coding sequence. For construction of the proKNAT4: GUS reporter construct, an approximately 6. 6-kb region of the sequence directly upstream of the KNAT4 coding sequence was amplified using BAC T5K6 as PCR template and cloned into pCRII-TOPO (Invitrogen). The KNAT4 upstream sequence was subcloned into the pRITA vector, which contains the GUS coding sequence and the terminator sequence from the nopaline synthase gene. For constitutive expression, the amiRNA sequences or the KNAT3 coding sequence were cloned into the ART7 vector, which contains the Cauliflower mosaic virus pro35S sequence and the terminator sequence from the octopine synthase gene. KNAT5, SAW2, and PNY coding sequences were amplified from Ler cDNA and cloned downstream of an Lac Op array [33] to generate responder cassettes used in the transcription activation system. All constructs were subcloned into pMLBART or pART27 binary vector and were introduced into Agrobacterium tumefaciens strain GV3001 by electroporation. Transgenic lines were generated by Agrobacterium-mediated transformation, and transformants were selected on soil on the basis of resistance to the BASTA or kanamycin. Primers used to clone the various cDNAs and promoters are described in S2 Table. Scanning electron microscopy was performed according to Alvarez and Smyth [71]. For light microscopy, cleared samples were prepared. Leaf samples were fixed overnight in 9: 1 (v: v) ethanol: acetic acid at room temperature. After rehydration in a graded ethanol series, samples were rinsed with water and were cleared with chloral hydrate solution [1: 8: 2 (v: w: v) glycerol: chloral hydrate: water]. For histochemical analysis of GUS activity, samples were infiltrated with GUS staining solution [0. 2% (w/v) Triton X-100,2 mM potassium ferricyanide, 2 mM potassium ferrocyanide, and 1. 9 mM 5-bromo-4-chloro-3-indolyl-β-glucuronide in 50 mM sodium phosphate buffer, pH 7. 0] and incubated at 37°C. Publically available KNOX and BELL coding nucleotide sequences representing taxa across land plants were manually aligned as amino acid translations using Se-Al v2. 0a11 (http: //tree. bio. ed. ac. uk/software/seal/). We excluded ambiguously aligned sequence to produce alignments for subsequent Bayesian analysis. Bayesian phylogenetic analysis was performed using Mr. Bayes 3. 2. 1 [72,73]. Three separate analyses were performed. The first included Chlorophyte algal and land plant KNOX sequences (S1 Fig.); the second included only land plant KNOX2 sequences (S2 Fig.); and the third included land plant BELL sequences (S17 Fig.). The fixed rate model option JTT + I was used based on analysis of the alignments with ProTest 2. 4 [74]. Sequence alignments and command files used to run the Bayesian phylogenetic analyses are provided upon request. | Title: Antagonistic Roles for KNOX1 and KNOX2 Genes in Patterning the Land Plant Body Plan Following an Ancient Gene Duplication Summary: Eukaryotes alternate between haploid (1n) and diploid (2n) stages during their life cycles, and often seen are remarkable differences in morphology and physiology between them. Land plants are multicellular in both generations, in contrast to their presumed ancestral green algae that develop multicellularity only in the haploid stage. TALE class homeodomain transcriptional factors play a key role in the activation of diploid development in diverse lineages of eukaryotes. A gene duplication event within this family in an ancestor of land plants had profound implications for land plant evolution. We show that the two subclasses resulting from the gene duplication event act to pattern, in a complementary manner, most above ground organs of the diploid stage of the flowering plant Arabidopsis. Their opposing activities sculpt the shape of leaves from entire to pinnate and control the architecture of the plant body, and thus providing plasticity for evolutionary tinkering. These results form a foundation for understanding how these genes have been co-opted from an ancestral role of regulating diploid gene expression in a zygote to directing sporophyte land plant body architecture and provide insight into the evolution of various forms of life cycles. | 9,707 | 295 | lay_plos | en |
Summarize: What Is an Endowment? A college or university's endowment fund—often referred to simply as an endowment—is an investment fund maintained for the benefit of the educational institution. Endowments may also hold cash or property. Income from the endowment is used to cover the cost of the college or university's operations and capital expenditures, to fund special projects, or for reinvestment. Typically, a college or university endowment includes hundreds, if not thousands, of individual funds that are the result of various agreements between donors and the recipient institution. There are several types of endowment funds. Donors may give funds to a true endowment, or permanent endowment. Oftentimes, donors impose restrictions on the institutions spending the principal balance of true endowments. Donors may also impose restrictions on the use of income earned on true endowments. True endowments may contain funds that the donors have dedicated to scholarships or faculty support, for example. A term endowment is an endowment where funds may be restricted by the donor for some period of time. After the set period of time has passed, unused funds or principal may become unrestricted. Institutions may also put other unrestricted funds, such as those from general gifts or bequests, in the institution's endowment. These funds are typically referred to as a quasi-endowment. Typically, when looking at the total value of an institution's endowments, true endowments, term endowments, and quasi-endowments are included. Of the $566.8 billion in endowment assets reported to NACUBO in 2017, $255.3 billion (45%) was in a true endowment, while $135.4 billion (24%) was in a quasi-endowment. Of the true endowment balance, $233.3 billion was donor restricted (41% of total endowment market value). The reported term endowment balance was $12.3 billion (2%). Tax Treatment of College and University Endowments Endowments are tax-exempt for one of two reasons. Either they are part of a university which is tax-exempt as a 501(c)(3) organization or a government entity (public universities), or the endowment itself has 501(c)(3) tax-exempt status. Contributions to 501(c)(3)s and government entities are generally tax deductible to the contributor under Internal Revenue Code (IRC) Section 170. Another benefit of an endowment's tax-exempt status is that, except for the recent excise tax on the net investment income of endowments at certain institutions, the investment earnings are tax free. The 501(c)(3) status of colleges and universities—and by extension their endowments—is a result of them being organized and operated exclusively for purposes listed in Section 501(c)(3), specifically charitable and educational purposes. If the return from endowments of colleges and universities were taxed currently at 35%, the revenue gain is estimated at $15.3 billion; taxed at the new corporate rate of 21%, the amount would be $9.9 billion. If only private universities and colleges were subject to a tax, the gain would be estimated at $10.4 billion at a 35% rate and $6.4 billion at a 21% rate, since public institutions are responsible for 32% of assets. This figure can be compared with the estimated value of other tax provisions that directly benefit educational institutions. These include the deduction for charitable contributions to educational institutions, estimated to reduce revenues by $10.5 billion in FY2017 (while the deduction is claimed by individuals and corporations, the benefit also accrues to institutions of higher education); private activity tax-exempt bonds for nonprofit educational institutions, estimated at $3.8 billion; and the share of general obligation tax exempt bonds benefits that accrue to public institutions of higher education, which are estimated at around $1.4 billion. These benefits total approximately $15.7 billion. Beginning in 2018, the value of these provisions would be expected to be somewhat smaller under the new tax rules. Excise Tax on Net Investment Income of Certain Institutions A 1.4% tax on net investment income is imposed annually on certain private colleges and universities beginning in tax years after December 31, 2017. For the purposes of the tax, net investment income is gross investment income and net capital gain, less expenses associated with earning that income. Gross investment income includes income from interest, dividends, rents, and royalties. To be subject to the tax, private colleges and universities must meet the following criteria: 1. have at least 500 tuition-paying full-time equivalent (FTE) students in the previous tax year (with more than 50% of these students located in the United States); and 2. have assets of at least $500,000 per student (excluding assets that are used directly in carrying out the institution's exempt educational purpose). Institutions that are subject to the tax and meet the above criteria are referred to as "applicable educational institution[s]." IRC Section 4968 is a new provision. Per the Conference Report accompanying P.L. 115-97, it is intended that the Secretary promulgate regulations to carry out the intent of the provision, including regulations that describe: (1) assets that are used directly in carrying out the educational institution's exempt purpose; (2) the computation of net investment income; and (3) assets that are intended or available for the use or benefit of the educational institution. The IRS has not yet released any guidance or regulations related to the new excise tax under IRC Section 4968. The Joint Committee on Taxation (JCT) estimated that the excise tax on net investment income of private colleges and universities with endowments of at least $500,000 per student would raise $1.8 billion in revenue over the 2018 through 2027 budget window. Annually, the tax is estimated to raise about $0.2 billion. Comparing the Tax Treatment of Endowments and Private Foundations Comparisons are often drawn between private foundations and college endowments, particularly when considering certain policy options (payout requirements, for example). Unlike private foundations, college endowments are not subject to a payout requirement. Private foundations are differentiated from tax-exempt public charities by their narrow bases of control and financial support. In order to limit accumulation of tax-exempt funds by foundations, Congress chose to require private foundations (and only private foundations) to pay out at least 5% of their fund every year under Internal Revenue Code (IRC) Section 4942. Private foundations are also subject to an excise tax on net investment income. The tax rate is 2%, but is reduced to 1% if the foundation's qualifying distributions exceed a historical average. When the tax on investment income of private foundations was enacted, Congress stated that the purpose of the tax was to have private foundations share in the cost of government oversight of the sector. Revenues from the tax, however, go to the general fund and are not earmarked for any specific purpose. College and University Endowments: Data Overview Endowment Balances There are several sources of data on endowment balances. Specifically, data on endowments can be obtained from the following sources: U.S. Department of Education : The U.S. Department of Education publishes data on endowment funds. Generally, information is provided on total endowment funds for all public and private colleges and universities and separately for the top 120 institutions. At the end of FY2015 (June 30, 2015), all institutions reported endowment funds of $547.2 billion. The 120 institutions with the largest endowments held $406.4 billion, or 74.3% of the total. National Association of College and University Business Officers (NACUBO) : NACUBO regularly gathers endowment data from a large number of public and private colleges and universities and affiliated foundations. At the end of FY2017 (June 30, 2017), 809 institutions reported $566.8 billion in endowment assets. The NACUBO survey provides the most up-to-date information on endowments, although it may not include the same institutions included in the U.S. Department of Education data. Internal Revenue Service : Data on endowments of 501(c)(3) (private) universities and colleges are reported on Schedule D of IRS Form 990 informational returns. IRS data representing 1,782 501(c)(3) higher education institutions reported $390.2 billion in endowment assets for 2014. Historically, the aggregate endowment values reported in the NACUBO survey have closely tracked the endowments data published by the Department of Education. While the Department of Education data includes all Title IV institutions, the NACUBO survey gathers information on endowments from some affiliated foundations in their survey. There have been fluctuations in endowment balances over time. Overall, endowment balances have increased substantially since the early 1990s, both in nominal and real (inflation-adjusted) terms ( Figure 1 shows trends in endowment balances since 1993, in inflation-adjusted terms). In 1993, endowment balances were $150.8 billion (inflation-adjusted to 2017 dollars). By 2015, endowment balances were $565.9 billion (inflation-adjusted). While the Department of Education has not yet released data for 2017, data from the NACUBO survey, which closely tracks Department of Education statistics on endowments, reported endowment assets of $566.8 billion for 2017. The share of endowments held by the top 120 institutions (the Department of Education regularly reports data on endowments held by the top 120 institutions) has remained roughly the same over time, at around 75%. Endowment values decreased substantially during the 2007-2008 financial crisis, and took several years to recover to precrisis levels. In 2007, endowments were valued at $484.4 billion (in inflation-adjusted 2017 dollars). Balances had declined to $372.0 billion (inflation-adjusted) by 2009. By 2013, inflation-adjusted endowment balances had reached $491.1 billion, before increasing further to $565.9 billion in 2015. College and University Endowments: FY2017 For the fiscal year ending June 30, 2017, data gathered by NACUBO from 809 universities and colleges reported total endowment assets of $566.8 billion. As illustrated in Table 1, endowment balances are heavily concentrated in a small share of institutions, with 12% of institutions holding 75% of endowments. Private nonprofit universities (classified as "independent" in Table 1 ) have a slightly higher share of endowments (68%) relative to their share of institutions (63%). Largest Private College and University Endowments In FY2017, according to NACUBO data, there were 31 private colleges and universities with endowments of at least $500,000 per student and 500 students (see Table 2 ). A list of the 100 largest college and university endowments, including public institutions can be found in Appendix B. A list of the 25 private institutions with the largest endowment per student, including institutions with less than 500 students, can be found in Appendix C. Harvard University had the largest endowment in FY2017, totaling more than $36.0 billion. On a per-student basis, Harvard's endowment was $1.5 million in FY2017. A total of five institutions had total endowment assets of $15.0 million or more, and a per-student endowment of at least $1.3 million (Harvard, Yale, Stanford, Princeton, and MIT). Endowment wealth is highly concentrated in these top institutions, with Harvard holding 6.4% of all endowment assets reported to NACUBO in FY2017. Yale held 4.8% of endowment assets in that same year, with Princeton holding 4.2% of endowment assets. Endowment assets are concentrated in private doctoral-granting universities. In FY2017, the average endowment per student at private doctoral-granting universities was $360,787 (the median was $74,767) (see Table 3 ). The average endowment per student at public doctoral-granting universities was $27,092 (with a median of $23,012). Payouts from College and University Endowments Spending from the endowment includes expenditures on student financial aid, faculty research, maintenance of facilities, and other campus operations. The spending rate, or payout rate, is this spending divided by the market value of the endowment at the beginning of the year (net of administrative expenses). Most institutions have a spending policy, where the payout rate is tied to a moving average of endowment value. Colleges and universities may deviate from predetermined spending policies, particularly in the face of negative financial shocks like the 2008-2009 financial crisis. NACUBO publishes data on average annual effective spending rates from endowment funds, or payouts. Since 1998, the average annual effective spending rate, or average payout rate, has fluctuated within a one-percentage-point band, hitting a period high of 5.1% in 2003 and period low of 4.2% in 2012 (see Figure 2 ). In 2017, the average payout rate was 4.4%. Spending (payout) rates also varied across different types of institutions in FY2017. Data presented in Table 4 show that the average payout rate was higher for private institutions (4.6%) when compared to public institutions (4.1%). The NACUBO data indicate that average payout rates in 2017 tended to increase with endowment size. The average payout rate was 4.8% for endowments with assets above $1 billion, with lower payout rates for smaller endowment size classes. Data on endowments of 501(c)(3) colleges and universities collected by the IRS can also be used to examine payout behavior. IRS data from 2014 indicate the average payout for 501(c)(3) institutions was 4.3% (see Table A-2 ). For institutions with large endowments (more than $1 billion in 2014), roughly half had payout rates between 3.8% and 4.9% in 2014. There is a wider distribution of payout rates across institutions with smaller endowments. Trends in payout rates over time have differed for institutions with large endowments as opposed to smaller endowments (see Figure 3 ). Payout rates generally trended downward between 2003 and 2008 (the average payout rate was 5.1% in 2003, as opposed to 4.3% in 2008). While average payouts trended upwards from 2008 through 2011, during that time, a gap developed between the average payout rate of institutions with endowments of $0.5 billion or more, and those with smaller endowments of less than $0.05 billion. In 2010, the average payout rate for institutions with endowments between $0.5 billion and $1 billion, and those with average endowments above $1 billion, was 5.7% and 5.6%, respectively. The average payout rate for institutions with an endowment between $0.025 billion and $0.05 billion was 4.1% in 2010, while the average payout rate for institutions with endowments of less than $0.025 billion was 3.5%. These trends suggest that during and in the period immediately following the Great Recession, institutions with larger endowments tended to increase payout rates. Institutions with large endowments, where spending from endowments funds a significant portion of operating expenses, tend to base payouts on average endowment values in recent years (often over a three-year period). Thus, payout rates tend to increase when endowments decline. Where payout rates tended to decline during and immediately following the Great Recession was among institutions with smaller endowments. As the economy has recovered, payout rates for institutions with large and smaller endowments have again converged and are close to the average across all institutions (4.4%). Endowment Fund Investments In recent years, on the whole, invested endowment assets have yielded strong returns. In FY2017, endowment assets included in the NACUBO survey earned a return of 12.2% on average, resulting in income of $64 billion. Returns have fluctuated over time (see Figure 4 ). In some years, FY2016 being the most recent example, average net returns have been negative. Returns on Endowment Fund Investments Endowment returns vary across different types of institutions and over time. In FY2017, institutions with larger endowments tended to earn higher returns. This pattern tends to hold when looking at returns over longer periods of time (3-, 5-, or 10-year average rates of return). Returns of public and private nonprofit (independent) institutions are similar (when looking at equal-weighted figures). Table 5 also provides information on the share of assets invested in alternative strategies (which includes hedge funds and private equity). Institutions with larger endowments tend to have a higher share of their endowment assets invested in alternative strategies. Institutions with smaller endowments tend to have most of their endowment assets invested in domestic equities, fixed income, or international equities. Trends in where endowment funds are invested over time are examined further below. Where Are Endowment Funds Invested? There has been a shift in where endowment assets are invested in recent years. Between 2002 and 2010, the share of endowment assets invested in equities declined from 50% to 31%. Since 2010, the share of endowment assets invested in equities increased to 36% (see Figure 5 ). The percentage of assets invested in fixed income declined from 23% to 8% over the 2002 to 2017 period. While the proportion of assets being invested in equity and fixed income declined, the share of assets invested in alternative strategies increased. The share of assets invested in alternative strategies, which includes hedge funds and private equity, increased from 20% in 2002 to 51% 2009. In 2017, the share of endowment assets invested in alternative strategies was 52%. Empirical research has explored why the asset allocation of university endowments has shifted towards alternative investments. Competition is one possible reason. There is evidence that institutions tend to increase the share of endowment holdings invested in hedge funds to "catch up" with schools that are competitors. Other research has observed that endowment managers are more likely to invest in alternative assets when institutions have higher and less variable background income. These institutions may be willing to take on the greater risk associated with alternative strategies investments. The shaded text box below provides additional background and information on UBIT and the use of blocker corporations to avoid UBIT. The increasing share of endowment assets invested or held in alternative strategies has raised concerns among some policymakers regarding the use of offshore "blocker" corporations to avoid the unrelated business income tax (UBIT) on hedge fund investments. This issue was the topic of a 2007 Senate Committee on Finance hearing and has continued to receive media attention. Policy Considerations and Options There are a number of policy options related to the tax treatment of endowments, should there be a desire to change the status quo. Policy options considered may depend on the overarching policy objective. For example, is the goal of the policy to encourage colleges and universities to use endowment funds for a specific purpose? Or is the objective of the policy to carve back or restrict the tax exemption currently provided to endowments? Or is the objective to look at endowments as a source of federal revenue? Identifying the goals of endowment-related tax policies may help inform the analysis of specific policy options. Leaving current-law tax treatment of endowments unchanged is also an option. Modify Tax on Endowment Net Investment Income Soon after the 1.4% excise tax on private college and university endowment's net investment income was enacted, legislation was introduced that would repeal the tax. Repeal of the tax is one option. Other policy options include those that would reduce or eliminate the tax in certain situations. For example, the tax could be reduced or eliminated for institutions that devote a certain portion of endowment returns to financial aid for low- and middle-income students. Alternatively, the tax could be waived for institutions that meet a fixed payout objective (discussed further below) or as part of a policy requiring increased reporting related to endowments and uses of endowment funds (discussed further below). Another option would be to repeal the tax, but require that some portion of endowment assets be managed by minority- and women-owned asset management firms. Options to modify or redesign the tax could also be considered. For example, the tax could be expanded to apply to more institutions, or the cut-off points (either the per-student or assets per FTE thresholds) changed. The tax could also be modified to be more punitive in nature. For example, Representative Reed has suggested an approach that would require institutions with endowment assets in excess of $1 billion to use at least 25% of investment gains to reduce the cost of attendance for lower- and middle-income students. Institutions not meeting this target would face a tax of 30% of net investment income, with the tax increasing for institutions that do not use endowments to reduce cost of attendance as stipulated over time. Taxing the net investment income of private college and university endowments had been proposed as part of an earlier tax reform effort. The Tax Reform Act of 2014 ( H.R. 1 ), a tax reform proposal introduced in the 113 th Congress by then Ways and Means Committee Chairman Dave Camp, proposed a flat excise tax for foundations and extended it to net investment earnings of private universities and colleges. Under this proposal, private colleges and universities with endowments in excess of $100,000 per student would be subject to a 1% excise tax. The tax on net investment returns of private college and university endowments enacted in the 2017 tax revision ( P.L. 115-97 ) applies to institutions with endowments in excess of $500,000 per student, at a rate of 1.4%. Additionally, the tax only applies to institutions with at least 500 students. Impose a Payout Requirement Some policymakers have proposed requiring endowments to pay out a minimum amount every year to prevent "an unreasonable accumulation of taxpayer-subsidized funds." One option would be to require that endowments have a minimum 5% payout rate, similar to that required of private foundations. In the face of rising tuition, it has been suggested that to achieve their charitable and educational objectives, colleges and universities should use a greater portion of their endowments to reduce college prices and make a college education more accessible and affordable for students. There are a number of policy design choices that could be considered when imposing a payout requirement. While a payout requirement of 5%—the payout requirement for private nonoperating foundations—is one option, some other level (higher or lower) could be chosen. The payout requirement could be restricted to certain endowments, such as those that exceed a certain threshold, either in absolute terms ($500 million, for example) or on a per-student basis. Payout requirements could be tied to investment earnings, or capped to not exceed investment earnings in down years. Payout requirements could also be determined on a rolling basis (5% over a 3-year period, for example). Payouts requirements could also be tied to tuition levels, metrics on student need (Pell grant recipients, for example), or students' receipt of federal student aid. Additional nuances could complicate various policy design choices. For example, if payout requirements were to be imposed in institutions with endowments per student above some predetermined level, what measure of students would be used (e.g., would the measure of students include graduate students; would the measure of students be fall enrollment, the 12-month unduplicated headcount, full-time equivalents, or some other measure?). Opponents of a payout requirement for endowments say the approach is misguided, and have been critical of various aspects of different payout policy options. When considering payout policies that target institutions with large endowments, some have suggested that these institutions may be more likely to offer robust financial aid and perhaps more likely to have modest tuition increases over time. In recent years, institutions with larger endowments have tended to have higher average payouts than institutions with smaller endowments. Thus, a payout requirement applied to all institutions regardless of endowment size could impose a greater burden on institutions with smaller endowments. Some have also questioned whether the 5% payout rate that currently applies to nonoperating foundations would be appropriate for endowments, noting that operating foundations often have lower effective payout rates. Finally, there are concerns that imposing a payout requirement might serve as a ceiling rather than a floor, leading institutions that would have paid out more than 5% (or whatever rate is required) to make payouts that meet the requirement but no more. One issue raised by university representatives is restrictions on endowments imposed by donors (i.e., donor-restricted funds, discussed earlier). Donor restrictions come in two forms (which often appear simultaneously): a requirement that the principal not be spent (so as to preserve the fund permanently) and a requirement that funds be spent for specified purposes. Mary Frances McCourt, representing NACUBO at a recent hearing, indicated that, in FY2014, colleges received $7.7 billion in new financial contributions to their endowments. Of those new gifts, 90% were restricted for a specific purpose by the donors. Since most endowments have grown over time, and proposed payout rates tend to be below earnings over time, restrictions on not spending principal could likely be designed to be manageable for most institutions. If a payout were to be required, payouts might come from quasi-endowments, as opposed to donor-restricted funds. As noted earlier in this report (see the " What Is an Endowment? " section), in 2017, 24% of all endowment funds were reportedly held in quasi-endowments, and 25% of endowment funds in a nondefined "other" category. Thus, across endowments as a whole, it could be possible to meet a payout requirement even if a sizable portion of endowment funds is restricted. What is not clear is how a payout requirement might affect specific institutions, where the proportion of restricted endowment funds may be higher. Restrictions on purposes are unlikely to impose a constraint to increased payouts, either for increasing spending generally or for the purpose of slowing tuition increases, because of the fungibility of money. That is, if some endowment funds are limited to specific purposes and increased spending for those purposes is not feasible (e.g., supporting an endowed chair), increased payouts from other endowments without restrictions or endowments that are devoted to student aid can be used to meet a payout requirement. Providing aid to students is one of the most common restrictions. Nevertheless, any legislation requiring a payout or a payout tied to a particular purpose might need a "safe harbor" so that colleges and universities would not be caught between legal restrictions on donations and payout requirements. Protections from fluctuations in asset values might also be addressed by requiring minimum payout averaged over several years. Tax Endowments or Impose Additional Taxes on Endowment Earnings Another option would be to impose a tax on the value of endowments or impose additional taxes on endowment earnings. As is the case with the payout option, there are a number of different ways such a policy could be designed. For example, the tax could only be applied to endowments of a certain size, or to institutions with "large" endowments that have increased tuition at a certain rate (more than the rate of inflation, for example). Endowment earnings could also be subject to tax. One option would be to impose a tax similar to the current maximum rate of 21% already imposed on tax-exempt entities for earnings from activities not related to their exempt purpose (the unrelated business income tax, UBIT). A tax on endowments or an additional tax on endowment earnings would generate additional federal revenues. These revenues could be earmarked to provide student aid across all colleges and universities, or could be treated as general fund revenues or used for other purposes. Whether a tax on endowments encourages universities to spend more is unclear: saving via the endowment becomes more expensive (as the after-tax return is lower) which would encourage payouts relative to endowment savings and accumulation. However, with lower after-tax returns, more saving may be needed to meet future endowment accumulation goals. Limit or Enhance Charitable Deductions for Gifts to Endowments Donors making contributions to endowments can claim the charitable deduction at the time the gift is made, even if the gift is not immediately used for charitable purposes. Gifts to endowments are often spent out over long periods of time. Reducing the value of the charitable deduction for gifts that are spent out over time, or are not immediately used for charitable purposes, could change incentives for giving donations that are related to endowments. Limiting the charitable deduction for restricted gifts to endowments or term endowments would reduce the tax incentive for making this kind of contribution. Taxpayers might choose instead to make nonrestricted contributions, substituting one form of giving for another. Since limiting the deduction would reduce the tax incentive for giving to endowments, overall contributions may fall. One option for implementing this approach could be to limit the deduction based on when the contribution is expected to be spent. For example, if the contribution is expected to support educational activities for 10 years, some adjustment could be made to reflect the fact that a dollar spent in the future is worth less than a dollar spent today, as a result of inflation. Since this type of adjustment could become complex, it could be limited to gifts of a certain size. Another option would be to provide an enhanced charitable deduction for certain types of gifts. For example, taxpayers making charitable donations of funds that would be used to offset tuition for lower- and middle-income students could receive an enhanced charitable deduction for such contributions. Change Policies for Certain Offshore Investments As discussed above, some have noted that the increased share of endowment assets being invested in alternative strategies, particularly hedge funds, raises concerns about the use of offshore blocker corporations to avoid UBIT. The ability to use offshore blocker corporations to avoid UBIT creates disparate tax treatment between debt-financed investments made domestically and those made offshore. Some have also expressed concern that current law creates an incentive to borrow to increase the level of the endowment, which could force spending cutbacks in a downturn. In 2007, then Chairman of the Ways and Means Committee Charles Rangel introduced a bill, H.R. 3970, which addressed the disparate tax treatment of domestic and offshore investments by exempting partnership income from the UBIT. Following hearings by the Senate Finance Committee in 2007, a draft proposal by Senators Grassley and Wyden circulated in August 2008 proposed instead to disallow the use of offshore blocker corporations by providing look-through rules to determine the source of earnings. Others have suggested that Congress could disallow the use of blocker corporations and expand the scope of UBIT rules covering debt-financed investment to apply to all forms of leveraged investment. Another option would be to repeal debt-financed UBIT rules, eliminating the incentive created under current law to use offshore blocker corporations. Increase Data Reporting In 2016, the Chairmen of the Senate Committee on Finance and the House Committee on Ways and Means, along with the Ways and Means Oversight Subcommittee Chairman, sent a letter to 56 colleges and universities requesting information regarding the operations and status of the institution's endowments. Others have suggested that the IRS Form 990 be modified to collect additional information on endowment funds. If endowment spending on financial aid and low- and moderate-income students is an issue of interest, it might be useful to collect additional information on spending on grants and scholarships. Another area of potential concern is administrative expenses associated with managing the endowment, including fees paid to third parties. It might also be helpful to policymakers to have additional information on the types of endowments held. For example, are endowments composed of quasi-endowments? Or is the use of assets otherwise restricted? Policymakers might also seek information on endowment holdings of real property. Collecting additional information on college and university endowments could increase transparency, while also being useful for policymakers in both policy and oversight capacities. A potential trade-off associated with collecting additional information is increased administrative burden. Appendix A. Endowment Data Reported to the IRS by 501(c)(3) Institutions Data on endowments of 501(c)(3) universities and colleges are reported on Schedule D of IRS Form 990 informational returns. The IRS makes available data files that contain a sample of information reported on the Form 990s. The most recent year for which IRS microdata files are available is 2014. Public universities and colleges are not required to file 990s and thus information on nonfiling institutions' endowments is not included in the IRS data. The IRS SOI 990 sample contains 1,048 observations of higher education entities, including colleges, universities, two-year colleges, and graduate and professional schools. Since the IRS SOI file is a sample, and not all smaller institutions are included, the sample weights can be used to estimate the population. Overall, this sample represents an estimated 1,782 institutions. For 2014, these institutions reported total endowments of $390.2 billion. In addition to collecting data on endowment balances, the IRS collects data on endowment contributions; net investment earnings, gains, and losses; grants and scholarships; other expenditures for facilities and programs; and administrative expenses. Contributions and positive net investment earnings increase endowment balances. Grants and scholarships, expenditures for facilities and programs, and administrative expenses reduce endowment balances. In 2014, $11.3 billion was reported in contributions to 501(c)(3) higher education endowments (see Table A-1 ). This amount is equal to 3.0% of the endowment balances reported at the beginning of 2014. Net investment earnings on endowment funds were reported to be $20.3 billion in 2012, or 5.4% of total endowment funds. The largest category of endowment spending in 2014 was for facilities and programs, $12.0 billion, or 3.2% of total endowment funds. Grants and scholarships were $4.2 billion in 2014, or 1.1% of endowment funds. Administrative costs were $1.0 billion, or 0.3% of endowment funds. Data reported by 501(c)(3) universities and colleges on their IRS Form 990 can be used to calculate payout rates. Following NACUBO, effective spending rates, or payout rates, are calculated as distribution for spending (spending on grants or scholarships and other expenditures for facilities and programs) divided by the beginning of year endowment value, net of administrative expenses. Payout rates for different endowment size classes are reported in Table A-2, in both dollar-weighted and equal-weighted terms. Analysis of the distribution of payout rate for 501(c)(3) higher education institutions reveals a couple of trends. First, median payouts tend to be higher for institutions with larger endowments. Second, payout rates for larger institutions are more concentrated. The difference between the payout rate for institutions at the 25 th percentile and those at the 50 th percentile is greater for institutions with smaller endowments. Appendix B. Top 100 College and University Endowments Table B-1 lists the top 100 U.S. college and university endowments reported to NACUBO as of FY2017, including both public and private institutions. Harvard University has the largest endowment, at $36.0 billion. Harvard held 6.4% of all endowment assets reported to NACUBO as of FY2017. The second-largest endowment was held by the Yale University system, at $27.2 billion, or 4.8% of all endowment assets. Taken together, the top 100 institutions listed in Table B-1 held 76.2% of endowment assets as of FY2017. Appendix C. Top 25 Private Institutions Ranked by Endowment Assets per Student Table C-1 includes the top 25 private institutions, ranked according to endowment assets per student. | Summary: Colleges and universities maintain endowments to directly support their activities as institutions of higher education. Endowments are typically investment funds, but may also consist of cash or property. Current tax law benefits endowments and the accumulation of endowment assets. Generally, endowment fund earnings are exempt from federal income tax. The 2017 tax revision (P.L. 115-97), however, imposes a new 1.4% excise tax on the net investment earnings of certain college and university endowments. Taxpayers making contributions to college and university endowment funds may be able to deduct the value of their contribution from income subject to tax. The purpose of this report is to provide background information on college and university endowments, and discuss various options for changing their tax treatment. This report uses data from the U.S. Department of Education, the National Association of College and University Business Officers (NACUBO) and Commonfund Institute, and the Internal Revenue Service to provide background information on college and university endowments. Key statistics, as discussed further within, include the following: In 2017, college and university endowment assets were $566.8 billion. Endowment assets have been growing, in real terms, since 2009. Endowment asset values fell during the 2007-2008 financial crisis, and took several years to fully recover. Endowment assets are concentrated, with 12% of institutions holding 75% of all endowment assets in 2017. Institutions with the largest endowments (Yale, Princeton, Harvard, and Stanford) each hold more than 4% of total endowment assets. The average spending (payout) rate from endowments in 2017 was 4.4%. Between 1998 and 2017, average payout rates have fluctuated between 4.2% and 5.1%. In recent years, institutions with larger endowments have tended to have higher payout rates. In 2017, endowment assets earned a rate of return of 12.2%, on average. Larger institutions tended to earn higher returns. Larger institutions also tended to have a larger share of assets invested in alternative strategies, including hedge funds and private equity. Changing the tax treatment of college and university endowments could be used to further various policy objectives. Current-law tax treatment could be modified to increase federal revenues. The tax treatment of college and university endowments could also be changed to encourage additional spending from endowments on specific purposes (tuition assistance, for example). Policy options discussed in this report include (1) a payout requirement, possibly similar to that imposed on private foundations, requiring a certain percentage of funds be paid out annually in support of charitable activities; (2) modifying the excise tax on endowment investment earnings; (3) a limitation on the charitable deduction for certain gifts to endowments; and (4) a change to the tax treatment of certain debt-financed investments in strategies often employed by endowments. | 8,742 | 691 | gov_report | en |
Summarize: By. James Daniel. and Ryan Gorman. Minnesota Vikings' star Adrian Peterson first encounter with his now-dead son came when he visited the dying boy at the hospital, it has been revealed. Having found out about the boy only months ago after the mother started having paternity tests to figure out the father, the footballer was planning a trip to see the child later this month. Sadly, his visit came much sooner and under much different circumstances than he envisioned. Mr Peterson skipped practice Friday to fly to South Dakota to see his battered son, in the hospital fighting for his life after Joseph. Robert Patterson, 27, the mother's boyfriend, was charged on Friday with aggravated assault and aggravated. assault on an infant. The two-year-old boy, named Ty, passed away on Friday at a South Dakota hospital after being. admitted on Wednesday. The football star's father Nelson confirmed that. the boy who died was his grandson. Charged: Joseph Robert Patterson, pictured in court today, left, is charged with aggravated battery of an infant and aggravated assault. It emerged in court that he had previously been charged over domestic violence. Requests for privacy: NFL star Adrian Peterson, pictured at a press conference today, acknowledged that he was dealing with a 'private matter' but said little else. It is believed that his'secret' son died today after being beaten by the boyfriend of the child's mother. First reported by TMZ, Mr Peterson never questioned being the child's father and offered to help any way he could. With plans to meet the boy later this month, it appears the former MVP was planning to take an active role in his son's life. Sadly, that involvement was limited to a hospital visit only hours before the boy was taken off life support and likely the upcoming funeral. Mr Peterson has never met Mr Patterson, sources told TMZ. The grieving father is now pushing for his toddler son's organs to be donated to people in need. The supposed killer appeared in court on Friday to be arraigned on the felony assault charges. Additional charges may be added now that the child has died, South. Dakota State’s Attorney's office said. The little boy was brutally beaten on Wednesday while in the violent man's home at Platinum Valley Apartments, Sioux Falls where he had recently moved with his mother. He was the sole caregiver at the time. Initially,. police were told that the child had choked on candy. Only later did it. emerge that his brain injuries were the result of being violently shaken. The arrest was made. after police found the little boy with injuries 'consistent with. abuse'. Mr Patterson is being held in Minnehaha County Jail, South Dakota on $750,000 cash bond and was scheduled to appear in court on October 23 but that will likely change in the wake of the toddler's death. South Dakota State’s Attorney Thomas Wollman filed motions on Friday ordering Mr Patterson to serve two year-long sentences that had been suspended on prior domestic assault charges. Mr Patterson had pleaded guilty in 2012 to simple assault in an incident involving an ex-girlfriend and her three-year-old son. The woman requested a protection order saying the vicious man had spanked her little boy so hard that she had to ice the welts on his buttocks, according to the Argus. She also said that he choked and punched her, threatened her with a knife. and held her in the bedroom against her will, according to records. The woman asked for the protection orders prior to the birth of the son she has with Mr Patterson. As part of his prior charges, Mr Patterson was ordered to undergo family violence training and to stay. away from the woman until completing it. A different woman requested a protection order against Patterson in 2004 in Jackson County. Remembered: Vanja Srdie shares a memory about Minnesota Vikings' Adrian Peterson's two-year-old son during a candlelight vigil at Sertoma Park in Sioux Falls. Family, friends and well-wishers gather to say goodbye to the little boy who was killed, allegedly by his mother's boyfriend. It is believed by officials that Mr Patterson only recently began a relationship with the mother of the child he allegedly killed. A candlelit vigil was held in Sertoma Park, Sioux Falls to remember the little boy on Friday. One woman told the Argus Leader that Ty 'was a spunky boy full of life, who was ''always running, jumping and into everything... but whose big brown eyes made it hard to be mad at him'. Another attendee, Vanja Srdic, 27, described the two-year-old as'very happy, very strong-willed, so much potential, a go-getter little boy. His mom was his world'. Following the news of his son's death, the NFL star tweeted: 'Thank you to my family, my fans and fans of other teams for their support. 'The NFL is a fraternity of brothers and I am thankful for the tweets, phone calls and text messages from my fellow players. Peterson added: 'God Bless everyone and thank u so much.' Peterson has been. pictured publicly with his son Adrian Jr, who is two, but it was another two-year-old boy who died on Friday. The football star also has a ten-year-old daughter named Adeja. Joseph Robert Patterson posted this picture on his Facebook page, believed to be him with his biological son. Patterson has been charged with the aggravated assault of his girlfriend's son on Friday. Joseph Patterson, who is charged with aggravated assault and aggravated battery on an infant, leaves the Lincoln County Courthouse on Friday. Other sports stars quickly offered their condolences following the child's tragic death. Basketball. star LeBron James tweeted: 'My deepest condolences goes out to Adrian. Peterson and his family! Sending prayers up for you homie! Nothing I can. say can help u through.'. Baseball player Bryce Harper wrote:. 'Prayers go out to @AdrianPeterson and his family! What a cruel world we. live in! Truly unbelievable! #GodBless' A friend of the boy's mother in Sioux Falls told CityPages. that the mother of the child had a casual relationship with the NFL. star when she lived in Twin Cities but had since moved to South. Dakota. A recent paternity. test revealed that Peterson was the little boy's father. The child has. his mothers' last name and the father is not identified on the birth. certificate. Peterson knew that he was the father of the child before he was tragically killed. In a phone interview with the Pioneer Press, Nelson Peterson said the injured boy is not Adrian Jr., but is his grandson. Minnesota Vikings player Adrian Peterson pictured this summer during a training camp with his two-year-old son Adrian Jr. Family: Adrian Peterson's two-year-old son Adrian Jr., lives at home with the footballer. 'All. I can say is, we are asking for prayers and for respect for our family. as we deal with this tragic situation,' Nelson Peterson said. Mr Peterson spoke to the media on Friday afternoon and thanked everyone for their support. He told reporters he was dealing with the situation and has asked for privacy. 'I really appreciate all the support that I’ve been receiving from fans, the Vikings organization,' he said. 'This. is a private matter and I would ask you all to please just respect my. privacy and not ask at all about the situation at hand. Thanks.' Patterson was charged with assault in 2012 and ordered to undergo family violence training. Asked. about his mindset, Mr Peterson said: 'Football is something I will always. fall back on. It gets me through tough times. Just being around the guys. in here, that’s what I need.' He said that he will be playing for the Minnesota Vikings on Sunday in a home game against the Carolina Panthers. However a Viking source told ESPN that following his son's death that 'could change'. Sioux Falls Police Department spokesman Sam Clemens said many people have asked about the identity of the boy's father. 'At. this point, we're not going to confirm anything. Who the father is does. not come into play in this investigation, so we’re not going to be. releasing any information as far as that goes,' he said. 'We're. not here to talk about the parents of this child,' said Mr Wollman. 'We wouldn't talk in a normal case about who. the parents are.' He then asked that people respect the family's privacy. 'The parents and extended family are suffering greatly.' Sioux Falls Police Department spokesman Sam Clemens said many people have asked about the identity of the boy's father. 'At this point, we're not going to confirm anything. Who the father is does not come into play in this investigation, so we’re not going to be releasing any information as far as that goes,' he said. Minnesota hosts the Carolina Panthers this weekend at the Metrodome and Peterson has insisted he will be fit and ready to play in the starting lineup | Summary: The NFL star only met his son for the first time, while he lie dying in bed on life support after the beating. Joseph Robert Patterson, 27, is. charged with aggravated assault on an infant - he has a history of. domestic violence. Additional charges are possible, according police. said on Friday. Two-year-old boy, named locally at Ty, died at a South Dakota hospital Friday morning after being taken off life support. The football player has another two-year-old son Adrian Jr. | 2,090 | 113 | cnn_dailymail | en |
Write a title and summarize: Chloroplast genomes in land plants contain approximately 100 genes, the majority of which reside in polycistronic transcription units derived from cyanobacterial operons. The expression of chloroplast genes is integrated into developmental programs underlying the differentiation of photosynthetic cells from non-photosynthetic progenitors. In C4 plants, the partitioning of photosynthesis between two cell types, bundle sheath and mesophyll, adds an additional layer of complexity. We used ribosome profiling and RNA-seq to generate a comprehensive description of chloroplast gene expression at four stages of chloroplast differentiation, as displayed along the maize seedling leaf blade. The rate of protein output of most genes increases early in development and declines once the photosynthetic apparatus is mature. The developmental dynamics of protein output fall into several patterns. Programmed changes in mRNA abundance make a strong contribution to the developmental shifts in protein output, but output is further adjusted by changes in translational efficiency. RNAs with prioritized translation early in development are largely involved in chloroplast gene expression, whereas those with prioritized translation in photosynthetic tissues are generally involved in photosynthesis. Differential gene expression in bundle sheath and mesophyll chloroplasts results primarily from differences in mRNA abundance, but differences in translational efficiency amplify mRNA-level effects in some instances. In most cases, rates of protein output approximate steady-state protein stoichiometries, implying a limited role for proteolysis in eliminating unassembled or damaged proteins under non-stress conditions. Tuned protein output results from gene-specific trade-offs between translational efficiency and mRNA abundance, both of which span a large dynamic range. Analysis of ribosome footprints at sites of RNA editing showed that the chloroplast translation machinery does not generally discriminate between edited and unedited RNAs. However, editing of ACG to AUG at the rpl2 start codon is essential for translation initiation, demonstrating that ACG does not serve as a start codon in maize chloroplasts. The evolution of chloroplasts from a cyanobacterial endosymbiont was accompanied by a massive transfer of bacterial genes to the nuclear genome, and by the integration of chloroplast processes into the host’s developmental and physiological programs [1]. In multicellular plants, chloroplasts differentiate from non-photosynthetic proplastids in concert with the differentiation of meristematic cells into photosynthetic leaf cells. This transformation is accompanied by a prodigious increase in the abundance of the proteins that make up the photosynthetic apparatus, which contribute more than half of the protein mass in photosynthetic leaf tissue [2]. Both nuclear and chloroplast genes contribute subunits to the multisubunit complexes that participate in photosynthesis. The expression of these two physically separated gene sets is coordinated by nucleus-encoded proteins that control chloroplast gene expression, and by signals emanating from chloroplasts that influence nuclear gene expression [1,3]. Beyond these general concepts, however, little is known about the mechanisms that coordinate chloroplast and nuclear gene expression in the context of the proplastid to chloroplast transition. Furthermore, a thorough description of the dynamics of chloroplast gene expression during this process is currently lacking. Despite roughly one billion years of evolution, the bacterial ancestry of the chloroplast genome is readily apparent in its gene organization and gene expression mechanisms. Most chloroplast genes in land plants are grouped into polycistronic transcription units [4] that are transcribed by a bacterial-type RNA polymerase [5] and translated by 70S ribosomes that strongly resemble bacterial ribosomes [6]. As in bacteria, chloroplast ribosomes bind mRNA at ribosome binding sites near start codons, sometimes with the assistance of a Shine-Dalgarno element [6]. Superimposed on this ancient scaffold are numerous features that arose post-endosymbiosis [7]. For example, a phage-type RNA polymerase collaborates with an RNA polymerase of cyanobacterial origin [5], and chloroplast RNAs are modified by RNA editing, RNA splicing, and other events that are either unusual or absent in bacteria [8]. Ribosome profiling data from E. coli revealed that the rate of protein output from genes encoding subunits of multisubunit complexes is proportional to subunit stoichiometry, and that proportional synthesis is typically achieved by differences in the translational efficiency of genes residing in the same operon [9,10]. As the majority of chloroplast gene products are components of multisubunit complexes, it is of interest to know whether similar themes apply. Furthermore, the gene content of polycistronic transcription units in chloroplasts has diverged from that in the cyanobacterial ancestor. Has “tuned” protein output been maintained in chloroplasts despite this disrupted operon organization? If so, what mechanisms achieve this tuning in light of the new gene arrangements and the new features of mRNA metabolism? In this work, we used ribosome profiling to address these and other questions of chloroplast gene regulation in the context of the proplastid to chloroplast transition. For this purpose, we took advantage of the natural developmental gradient of the maize seedling leaf blade, where cells and plastids at increasing stages of photosynthetic differentiation form a developmental gradient from base to tip [11]. By using the normalized abundance of ribosome footprints as a proxy for rates of protein synthesis, we show that the rate of protein output from many chloroplast genes is tuned to protein stoichiometry, and that tuned protein output is achieved through gene-specific balancing of mRNA abundance with translational efficiency. This comprehensive analysis revealed developmentally programmed changes in translational efficiencies, which superimpose on programmed changes in mRNA abundance to shift the balance of protein output as chloroplast development proceeds. We analyzed tissues from the same genetic background and developmental stage as used in previous proteome [2] and nuclear transcriptome [12,13] studies of photosynthetic differentiation in maize. Four leaf sections were harvested from the third leaf to emerge in 9-day old seedlings (Fig 1A): the leaf base (segment 1), which harbors non-photosynthetic proplastids; 3–4 cm above the base (segment 4), representing the sink-source transition and a region of active chloroplast biogenesis; 8–9 cm above the base (segment 9), representing young chloroplasts; and a section near the tip (segment 14) harboring mature bundle sheath and mesophyll chloroplasts [2,12]. The developmental transitions represented by these fractions are illustrated in the immunoblot assays shown in Fig 1B. The mitochondrial protein Atp6 is most abundant in the two basal sections, subunits of photosynthetic complexes (AtpB, PetD, PsaD, PsbA, NdhH, RbcL) are most abundant in the two apical sections, and a chloroplast ribosomal protein (Rpl2) exhibits peak abundance in the two middle sections. These developmental profiles are consistent with prior proteome data [2]. To explore the contribution of differential chloroplast gene expression to the distinct proteomes in bundle sheath and mesophyll cells, we also analyzed bundle sheath and mesophyll-enriched fractions from the apical region of seedling leaves. Standard protocols for the separation of bundle sheath and mesophyll cells involve lengthy incubations that are likely to cause changes in ribosome position. We used a rapid mechanical fractionation method that minimizes the time between tissue disruption and the generation of ribosome footprints (see Materials and Methods). Markers for each cell type were enriched 5- to 10-fold in the corresponding fraction (Fig 1C). This degree of enrichment is comparable to that of the fractions used to define mesophyll and bundle sheath-enriched proteomes in maize [14]. We modified our previous method for preparing ribosome footprints from maize leaf tissue [15] to reduce the amount of time and tissue required, and to reduce contamination by non-ribosomal ribonucleoprotein particles (RNPs). In brief, leaf tissue was flash frozen and ground in liquid N2, thawed in a standard polysome extraction buffer, and treated with Ribonuclease I to liberate monosomes. Ribosomes were purified by pelleting through a sucrose cushion under conditions that leave chloroplast group II intron RNPs (~600 kDa) [16] in the supernatant (S1A Fig). RNAs between approximately 20 and 35 nucleotides (nt) were gel purified and converted to a sequencing library with a commercial small RNA library kit that has minimal ligation bias [17]. rRNA contaminants were depleted after first strand cDNA synthesis by hybridization to biotinylated oligonucleotides designed to match abundant contaminants detected in pilot experiments (S1 Table). Approximately 35 million reads were obtained for each “Ribo-seq” replicate, roughly 50% of which aligned to mRNA (S2 Table). RNA-seq data was generated from RNA extracted from aliquots of each lysate taken prior to addition of RNAse I. Replicate RNA-seq and Ribo-seq assays showed high reproducibility (Pearson correlation of >0. 98, S2 Fig). Almost all plastid genes were represented by at least 100 reads per replicate in all datasets (S3 Fig). Several clusters of low abundance reads mapped to small unannotated ORFs, but further investigation is required to evaluate which, if any, of these are the footprints of translating ribosomes. Ribosomes in the cytosol, mitochondria, and chloroplasts have distinct genetic origins. Accordingly, the ribosome footprints from each compartment displayed different size distributions (Fig 2A). The cytosolic ribosome footprints showed a minor peak at 23 nucleotides and a major peak at 31 nucleotides, similar to observations in yeast [18]. The mitochondrial data showed a major peak at 28–29 nucleotides and a minor peak at 36 nucleotides, similar to the 27 and 33-nt peaks reported for human mitochondria [19]. The plastid ribosome footprints had a broad size distribution suggestive of two populations, with peaks at approximately 30 and 35 nucleotides. A similar distribution was observed in pilot experiments involving the gel purification of RNAs up to 40-nt (S1B Fig) indicating that the peak at 35-nt was not an artifact of our gel purification strategy. A broad and bimodal size distribution was also observed for chloroplast ribosome footprints from the single-celled alga Chlamydomonas reinhardtii, albeit with peaks at slightly different positions [20]. The two prior reports of ribosome footprint size distributions in plants [21,22] did not parse the data from the three compartments, but the 31-nucleotide modal size reported in those studies is consistent with our data. Our data show the 3-nucleotide periodicity expected for ribosome footprints (Fig 2B and 2C). Interestingly, the degree of periodicity varies with footprint size (S4 Fig). The reads are largely restricted to open reading frames in the cytosol (Fig 2C) and chloroplast (Fig 2D). Taken together, these results provide strong evidence that the vast majority of the Ribo-seq reads come from bona-fide ribosome footprints. The placement of ribosome P and A sites with respect to ribosome footprint termini has not been reported for any organellar ribosomes or for cytosolic ribosomes in maize. A meta analysis of our data showed that the position of the 3’ end of ribosome footprints from initiating and terminating ribosomes in chloroplasts and mitochondria is constant with respect to start and stop codons, respectively, regardless of footprint size; however, the position of the 5’ ends varies with footprint size (Fig 2E, S4C Fig). Therefore, the positions of the A and P sites in organellar ribosomes can be inferred based on the 3’-ends of their footprints, as is also true for bacterial ribosomes [23,24]. The modal distance between the start of the P site in chloroplast ribosomes and the 3’-ends of chloroplast ribosome footprints is 7 nucleotides. By contrast, cytosolic ribosome footprints are approximately centered on the P site regardless of footprint size (S4B Fig). The partitioning of ribosome footprints among the three genetic compartments shifts dramatically during the course of leaf development (Fig 2G). The contribution of cytosolic translation drops from 99% at the leaf base to 57% in the apical leaf sections due to the increasing contribution of ribosome footprints from chloroplasts. This shift of cellular resources towards chloroplast translation corresponds with the massive increase in the content of photosynthetic complexes harboring plastid-encoded subunits (Rubisco, PSII, PSI, cytochrome b6f, ATP synthase, NDH) (Fig 1). Ribosome footprints from mitochondria accounted for a very small fraction of the total at all stages. However, our protocol was not optimized for the quantitative recovery of mitochondrial ribosomes so these data may not reflect the total mitochondrial ribosome population. In the discussion below we define the “translational output” of a gene as the abundance of ribosome footprints per kb per million reads mapped to nuclear coding sequences (RPKM), and we use this value to compare rates of protein synthesis among genes on a molar basis. This is a typical interpretation of Ribo-seq data, and it is based on evidence that the bulk rate of translation elongation on all ORFs is similar under any particular condition, despite the fact that ribosome pausing can lead to the over-representation of ribosomes at specific positions [9,25]. Although this may be an over simplification in some instances, this interpretation of our data produced results that are generally coherent with current understanding of chloroplast biogenesis (see below). Group II introns interrupt eight protein-coding genes in maize chloroplasts. These present a challenge for data analysis because the unspliced transcripts make up a substantial fraction of the RNA pool [16] and translation can initiate on unspliced RNAs and terminate within introns [15]. We therefore calculated translational output based solely on the last exon (normalized to exon length). Data summaries presented below include RNA-seq data only for that subset of intron-containing genes for which multiple methods of analysis provided consistent values for the abundance of spliced RNA isoforms (see Materials and Methods). Fig 3 summarizes the abundance of Ribo-seq and RNA-seq reads from protein-coding chloroplast genes in each of the four leaf segments. To display the low values from Segment 1, they are replotted with a smaller Y-axis scale in S5 Fig. The abundance of mRNA from genes in the same transcription unit (Fig 3A and S5A Fig, bracketed arrows) is typically similar, but the protein output of co-transcribed genes varies considerably. Translational efficiency (translational output /mRNA abundance) varies widely among genes (Fig 3A and S5A Fig, bottom). The atpH mRNA is the most efficiently translated of any chloroplast mRNA at all four developmental stages, surpassing even psbA, whose product is the most rapidly synthesized protein in photosynthetic tissues [26]. Prodigious psbA expression results from very high mRNA abundance in combination with a translational efficiency that is comparable to that of other photosystem genes. When the data are grouped according to gene function, correlations between function and translational output become apparent (Fig 3B). For example, the translational output of genes encoding subunits of ribosomes and the NDH complex are consistently very low, whereas the translational output of genes encoding subunits of PSI, PSII, the ATP synthase, and the cytochrome b6f complex are consistently much higher. These trends mirror the abundance of these complexes as inferred from proteome data [27]. The data for complexes whose subunits are not found in a 1: 1 ratio show further that translational output is tuned to subunit stoichiometry. For example, the chloroplast-encoded subunits of the ATP synthase (AtpA, AtpB, AtpE, AtpF, AtpH, AtpI) are found in a 3: 3: 1: 1: 14: 1 molar ratio in the complex [28,29]. The translational output of their genes mirrors this stoichiometry quite well, whereas mRNA abundance does not (Fig 4A). These genes are distributed between two transcription units (Fig 4A). A single mRNA encodes AtpB and AtpE, whose rates of synthesis are tuned via differences in translational efficiency. The atpI-atpH-atpF-atpA primary transcript is processed to yield various smaller isoforms [30] but the abundance of RNA from each gene is nonetheless quite similar (Fig 4A). The translational output of the atpH gene is boosted relative to that of its neighbors primarily through exceptionally high translational efficiency (Fig 4A bottom). In a second example, the unequal stoichiometry of subunits of the plastid-encoded RNA polymerase (PEP) (2 RpoA: 1 RpoB: 1 RpoC1: 1 RpoC2) [5] is mirrored by the relative translational output of the corresponding genes (Fig 4B). In this case, however, tuning occurs primarily at the level of mRNA accumulation. The plastid-encoded subunits of PSI, PSII, the cytochrome b6f complex, the NDH complex, and chloroplast ribosomes are found in equal numbers in their respective complex. Genes encoding subunits of each of these complexes are distributed across multiple transcription units, many of which also encode subunits of other complexes. This gene organization sometimes results in considerable disparity in mRNA level among subunits of the same complex (Fig 3B bottom). In general, such differences are buffered by opposing changes in translational efficiency, such that translational outputs more closely reflect protein stoichiometry than does mRNA abundance (see, for example, the NDH complex in S6B Fig). In the case of PSI (Fig 4C), the structural genes (psaA, psaB, psaC, psaJ, psaI) exhibit an approximately three-fold range of translational output, but all of these genes vastly out produce two genes encoding PSI assembly factors (ycf3 and ycf4) [31–33]. The psaI and ycf4 genes are adjacent in the same polycistronic transcription unit (Fig 4C bottom), and their difference in translational output is programmed primarily by a difference in translational efficiency. The translational output of psbN, which encodes a PSII assembly factor [34], is likewise much less than that of structural genes for PSII (Fig 4D). Taken together, this body of data shows that the tuning of translational output to protein stoichiometries is accomplished via trade-offs between mRNA level and translational efficiency, with this balance differing from one gene to the next. Where mRNA abundance closely matches protein stoichiometry, differences in translational efficiency make only a small contribution (as observed for rpoA, rpoB, rpoC1 and rpoC2). Where mRNAs are severely out of balance with protein stoichiometry, differences in translational efficiency compensate. The translational output of PSII structural genes is well matched, with the notable exception of psbA (Fig 4D), whose output vastly exceeds that of other genes in photosynthetic leaf segments (segments 9 and 14). This behavior is consistent with the known properties of the psbA gene product, whose damage and rapid turnover during active photosynthesis is compensated by a high rate of synthesis to support PSII repair [26]. Setting psbA aside, the relative translational outputs of other genes only approximate the stoichiometries of their products: several-fold differences between relative output and stoichiometry are common among subunits of a particular complex, suggesting that proteolysis of unassembled subunits serves to fine-tune protein stoichiometries. It is also possible that the calculated translational outputs do not perfectly reflect rates of protein synthesis due to differences in translation elongation rates among mRNAs. That said, instances in which translational outputs are particularly discordant among subunits of the same complex are worthy of note, as this may reflect physiologically relevant behaviors. For example, the translational output of ndhK is balanced with other ndh genes in non-photosynthetic leaf segments but ndhK substantially out produces the other ndh genes in mature chloroplasts (S6B Fig). This behavior is reminiscent of psbA, and suggests that NdhK may be damaged and replaced during active photosynthesis. To explore the dynamics of chloroplast gene expression during the proplastid to chloroplast transition, we calculated standardized values for translational output, mRNA abundance and translational efficiency such that developmental shifts can be compared despite large differences in signal magnitude. This analysis shows that the developmental dynamics of translational output varies widely among genes (Fig 5A top). The standardized values were used as the input for hierarchical clustering, which produced four clusters from the translational output data, four from the mRNA data, and five from the translational efficiency data (Fig 5B, S7 Fig). The genes in each cluster are identified by color in Fig 5A. Although the transitions between clusters are not marked by obvious distinctions, the distinct trends defining each cluster are clear in the plots in Fig 5B. Genes whose translational output and mRNA abundance peak early in development (segment 4) generally encode components of the chloroplast gene expression machinery (rpl, rps, rpo, matK) (Fig 5A and 5C). Most genes encoding components of the photosynthetic apparatus (psb, psa, atp, pet genes) have peak mRNA and translational output in young chloroplasts (segment 9). A handful of photosynthesis genes either maintain or increase translational output and mRNA in mature chloroplasts (segment 14) (Fig 5A and 5C). There is considerable similarity among the clusters produced from the translational output and mRNA data (Fig 5A and 5B), implying that programmed changes in mRNA abundance underlie the majority of developmental shifts in translational output. However, changes in translational efficiency also influence the developmental shifts in translational output (Fig 5A bottom). In general, ORFs encoding proteins involved in photosynthesis are more efficiently translated later in development and those encoding gene expression factors are more efficiently translated early in development, albeit with numerous exceptions (Fig 5A bottom, 5C right). Transcription units that encode both photosynthesis and gene expression factors provide revealing examples of distinct translational dynamics. In the psaA-psaB-rps14 transcription unit, for example, rps14 is found in a translational output cluster with other genes involved in gene expression, whereas psaA and psaB reside in a translational output cluster with other photosynthesis genes (Fig 5A top). This results from distinct developmental shifts in translational efficiency: the rps14 ORF is translated more efficiently early in development whereas psaA and psaB are more efficiently translated later in development (Fig 5D). The psaI-ycf4-cemA-petA transcription unit provides a second example. The translational output of psaI, cemA, and petA show similar developmental dynamics, but ycf4 clusters with different genes due to more efficient translation earlier in development (Fig 5D). Again, these distinct patterns correlate with function, as psaI and petA encode components of the photosynthetic apparatus, whereas ycf4 encodes an assembly factor for PSI [31,32]. Many polycistronic RNAs in chloroplasts are processed to smaller isoforms. Although the impact of processing on translational efficiencies remains unclear [35,36], it is plausible that programmed changes in the accumulation of processed isoforms could uncouple the expression of cotranscribed genes during development. To address this possibility, we used RNA gel blot hybridization to analyze transcripts from two transcription units that include genes whose translational efficiencies exhibit distinct developmental dynamics: psaI-ycf4-cemA-petA and psaA-psaB-rps14 transcription units (S8 Fig). Processed rps14-specific transcripts accumulate preferentially in immature chloroplasts (segment 4), correlating with the stage at which rps14 is most efficiently translated. Analogously, a monocistronic psaI isoform accumulates preferentially in segments 4 and 9 where psaI is most efficiently translated. Various cause and effect relationships may underlie these correlations, as is discussed below. In maize and other C4 plants, photosynthesis is partitioned between mesophyll (M) and bundle sheath (BS) cells. Three protein complexes that include plastid-encoded subunits accumulate differentially in the two cell types: Rubisco and the NDH complex are enriched in BS cells whereas PSII is enriched in M cells [2,14]. Differential accumulation of several chloroplast mRNAs in the two cell types has been reported [37–41], but a comprehensive comparison of chloroplast gene expression in BS and M cells has been lacking. To address this issue we performed RNA-seq and Ribo-seq analyses of BS- and M- enriched leaf fractions. The translational output of genes encoding subunits of Rubisco, PSII, and the NDH complex (Fig 6A) correlated well with the relative abundance of subunits of these complexes in the same sample preparations (Fig 1C), and with quantitative proteome data [2]. Cell-type specific differences in mRNA accumulation (Fig 6B) can account for many of the differences in translational output (Fig 6A), indicating that differences in transcription and/or RNA stability make a strong contribution to preferential gene expression in one cell type or the other. However, the data suggest that differences in translational efficiency contribute in certain instances (Fig 6C). Four genes encoding PSII core subunits (psbA, psbB, psbC, psbD) provide the most compelling examples, as their translational output is considerably more biased toward M cells than are their mRNA levels. Organellar RNAs in land plants are often modified by an editing process that converts specific cytidine residues to uridine [42,43]. Some sites are inefficiently edited, which raises the question of whether the translation machinery discriminates between edited and unedited RNAs. The protein products of several unedited mitochondrial RNAs have been detected in plants [44,45]. We used our Ribo-seq and RNA-seq data to examine this issue for chloroplast RNAs. Fig 7 summarizes the data for those sites of editing that are represented by at least 100 reads in both the Ribo-seq and RNA-seq data in at least two replicates (17 of the 28 edited sites in the maize chloroplast transcriptome). In general, the percent editing was similar in the RNA-seq and Ribo-seq data, implying little discrimination between edited and unedited RNAs by the translation machinery. There were, however, two major exceptions: rpl2 (nt 2) and ndhA (nt 563). In these cases a large fraction of the RNA-seq reads came from unedited RNA, whereas virtually all of the Ribo-seq reads came from edited sites. These two sites have unusual features that can account for the preferential translation of the edited RNAs. Editing at the ndhA site is linked to the splicing of the group II intron in the ndhA pre-mRNA: the site is not edited in unspliced transcripts and it is fully edited in spliced transcripts [46–48]. Failure to edit unspliced RNA is presumably due to the position of the intron between the edited site and the cis-element that specifies it. Translation that initiates on unspliced ndhA RNA would terminate at an in-frame stop codon within the intron. Thus, exon 2 is translated only from spliced RNAs, and these are 100% edited. In the case of rpl2, the editing event creates an AUG start codon from an ACG precursor; this is the only editing event in maize chloroplasts that creates a canonical start codon. Although it has been reported that ACG can function as a start codon in chloroplasts [49,50], our data show that this particular ACG is strongly discriminated against by initiating ribosomes. The fact that the Ribo-seq data show the expected strong bias toward edited rpl2 and ndhA (563) instills confidence that valid conclusions can be made from our data for other edited sites. Approximately 40% of the petB and ndhA (nt 50) sequences are unedited in both the RNA-seq and Ribo-seq data, indicating that these unedited sequences give rise to a considerable fraction of the translational output of the corresponding genes. Editing of the petB site is essential for the function of its gene product (cytochrome b6) [51]. It seems likely that the product of this unedited RNA is either unstable or selected against during complex assembly, as has also been suggested for the products of two unedited transcripts in mitochondria [52,53]. The remaining sites show almost complete editing in the RNA-seq data and, as expected, in the Ribo-seq data as well. That said, there is an overall trend toward less representation of unedited sequences in the Ribo-seq data than in the RNA-seq data. This may simply be a kinetic effect as would be expected if ribosome binding is slow in comparison to editing, such that ribosomes generally translate older (and therefore more highly edited) mRNAs. Ribosome profiling data from bacteria revealed a striking correspondence between the stoichiometry of subunits of multisubunit complexes and their relative rates of synthesis [9,10]. Our results show that the relative translational outputs of chloroplast genes likewise approximate the relative abundance of the gene products. This tuning is apparent when comparing sets of genes encoding different complexes (e. g. compare genes encoding the low abundance NDH complex to genes encoding the highly abundant PSI and PSII complexes) (Fig 3B), and when comparing genes encoding subunits of the same complex (e. g. the PEP RNA polymerase and the ATP synthase) (Fig 4A and 4B). Our calculations of translational output rest on the assumption that the rate of translation elongation on all mRNAs is similar under any particular condition. This same assumption produced remarkable concordance between protein stoichiometry and inferred translational output in bacteria [9,10]. Although our results show a clear trend toward “proportional synthesis”, they also suggest that the tuning of protein output to stoichiometry is less precise in chloroplasts than it is in bacteria. Subunits of photosynthetic complexes are subject to proteolysis when their assembly is disrupted [55], and a similar (albeit wasteful) mechanism could contribute to balancing stoichiometries when proteins are synthesized in excess under normal conditions. That said, instances in which inferred translational outputs are particularly incongruent with protein stoichiometries may reflect physiologically informative behaviors. The most prominent examples of “over-produced” proteins in our data are PsbA and PsbJ in PSII, PsaC and PsaJ in PSI, NdhK in the NDH complex, Rps14 in ribosomes, and PetD, PetL and PetN in the cytochrome b6f complex (Fig 4 and S6 Fig). Disproportionate synthesis of PsbA is well known, and compensates for its damage and proteolysis during photosynthesis [26]. The other proteins suggested by our data to be produced in excess may likewise be subject to more rapid turnover than their partners in the assembled complex. A proteomic study in barley demonstrated that subunits of each photosynthetic complex generally turn over at similar rates [56], but data for these particular proteins were not reported. Interestingly, the inferred rates of synthesis of PsbA, PetD, and NdhK are well matched to those of their partner subunits early in development, but outpace those of their partners in mature chloroplasts (Fig 4D, S6 Fig). This feature of psbA expression coincides with the need to replace its gene product, D1, following photo-induced damage and proteolysis [26]. By extension, the developmental dynamics of petD and ndhK expression suggest that their gene products may turn over more rapidly than their partners as a consequence of photosynthetic activity. In bacteria, proportional synthesis of subunits within a complex is achieved largely through the tuning of translational efficiencies among ORFs on the same mRNA [9,10]. In chloroplasts, genes encoding subunits of the same complex are generally distributed among multiple transcription units [4] and RNA segments within a transcription unit often accumulate to different levels [8]. It is interesting to consider how this shift in the gene expression landscape is reflected in the mechanisms that balance protein output among genes. In the case of the four genes encoding the PEP RNA polymerase, relative translational outputs closely match the 2: 1: 1: 1 protein stoichiometry, and this is programmed primarily at the level of mRNA abundance (Fig 4B). By contrast, widely varying translational efficiencies are superimposed on small variations in mRNA abundance to tune translational output to protein stoichiometry in the ATP synthase complex (Fig 4A). Genes for ribosomal proteins are distributed among ten transcription units, several of which also encode proteins involved in photosynthesis (see Fig 3A). For example, rps14 is cotranscribed with genes encoding the reaction center proteins of PSI (psaA/psaB), and translational outputs within this transcription unit are balanced by large differences in translational efficiency (Fig 4C). Similarly, the psaI transcription unit encodes subunits of the abundant PSI and cytochrome b6f complexes, a low abundance PSI assembly factor (Ycf4) and a protein of unknown function (CemA); large differences in translational efficiency adjust the translational outputs to meet these different needs (Fig 4C bottom). For complexes harboring plastid-encoded subunits in equal stoichiometries (ribosomes, NDH, PSI, PSII, cytochrome b6f), compensating differences in translational efficiency generally buffer differences in mRNA level. Taken together, these results imply that mRNA abundance and translational efficiencies have coevolved in chloroplasts to produce proteins in close to the optimal amounts. In some instances, mRNA levels are sharply out of balance with protein stoichiometries, in which case differential translational efficiencies compensate. In other instances, mRNA levels approximate protein stoichiometries, and translational efficiencies are similar. These observations further suggest that for most genes in maize chloroplasts, mRNA levels and translational efficiencies are poised such that they limit the rate of protein synthesis to a similar extent. This view is further supported by the developmental dynamics discussed below. In Chlamydomonas chloroplasts, synthesis of subunits within the same photosynthetic complex is coordinated through assembly-dependent auto-regulatory mechanisms [57]. By contrast, current data for angiosperm chloroplasts suggest that translational efficiencies are generally independent of the assembly status of the gene products [15,58]. It seems likely that translational efficiencies are dictated by the interplay between the sequence and structure of RNA proximal to start codons and the proteins that bind this region. Translation initiation in chloroplasts sometimes involves a Shine-Dalgarno interaction and is facilitated by an unstructured translation initiation region [6,59]. Additionally, the translation of some chloroplast ORFs requires the participation of gene-specific translation activators [15,60–75]. Such proteins provide a means for tuning protein synthesis within and between transcription units. The atpH ORF and its nucleus-encoded translational activator PPR10 exemplify this mechanism. The exceptionally high translational efficiency of atpH (Fig 3A) boosts its translational output to match the high stoichiometry of AtpH in the ATP synthase complex (Fig 4A); this high translational efficiency requires the binding of PPR10 adjacent to the atpH ribosome binding site, an interaction that prevents the formation of inhibitory RNA structures involving the translation initiation region [15,30,62]. Our results provide a comprehensive view of the dynamics of chloroplast mRNA abundance and translation during the proplastid to chloroplast transition. The majority of genes involved in chloroplast gene expression exhibit peak mRNA abundance and translational output in developing chloroplasts (segment 4) whereas the majority of genes encoding subunits of the photosynthetic apparatus exhibit peak mRNA abundance and translational output in young chloroplasts (segment 9) (Fig 5C). That said, even in proplastids (segment 1), genes involved in photosynthesis are generally represented by more mRNA and a higher translational output than are those involved in chloroplast gene expression (S5 Fig). Our data show that programmed changes in translational efficiency combine with changes in mRNA abundance to produce developmental shifts in translational output (Fig 5A). In general, translational efficiency is lowest at the leaf base, reflecting the low ribosome content in proplastids. The translational efficiency of most ORFs peaks in young chloroplasts (segment 9). In this context, it is intriguing that one subset of genes exhibit peak translational efficiency in the basal leaf segments (Fig 5A, bottom; Fig 5C, right), whereas another subset increases in translational efficiency right out to the leaf tip (Fig 5A and 5C). The former group is strongly enriched for “biogenesis” genes (RNA polymerase, ribosomes, assembly factors), and the latter for photosynthesis genes. Possible mechanisms underlying these distinct “translational regulons” are discussed below. A study in Chlamydomonas showed that changes in chloroplast mRNA abundance are not reflected by corresponding changes in rates of protein synthesis, leading to the conclusion that translation is the primary rate-limiting step [76]. The data presented here suggest that this is not the case in maize chloroplasts. The developmental shifts in mRNA abundance were largely mirrored by shifts in translational output (Fig 5A and 5B), implying that mRNA abundance has considerable impact on the output of most chloroplast genes in maize. Likewise, chloroplast DNA copy number limits gene expression in developing maize chloroplasts [77] but does not limit gene expression in Chlamydomonas chloroplasts [76]. It is perhaps unsurprising that mechanisms of gene regulation have diverged in the chloroplasts of vascular plants and single-celled algae, given their very different developmental and ecological contexts. Our data revealed a strong correlation between gene function and the developmental dynamics of mRNA abundance (Fig 5C middle): mRNAs encoding proteins involved in gene expression generally peak in abundance earlier in development than do those encoding components of the photosynthetic apparatus. This finding was foreshadowed by analyses of several chloroplast mRNAs during leaf development in barley and Arabidopsis [78–80]. Land plant chloroplasts harbor two types of RNA polymerase, a single-subunit nucleus-encoded polymerase (NEP) and a bacterial-type plastid-encoded polymerase (PEP) [5]. The ratio of NEP to PEP drops precipitously during chloroplast development, and this likely makes a large contribution to the changes in chloroplast mRNA pools [5,78,80,81]. There is evidence that NEP plays an especially important role in the transcription of “house keeping” genes, and PEP in the transcription of photosynthesis genes [5]; however, most chloroplast genes can be transcribed by both NEP and PEP [82], and the degree to which each polymerase contributes to the transcription of each gene during the course of chloroplast development remains unknown. Chloroplasts harbor several nucleus-encoded sigma factors that target PEP to distinct promoters [83], and these provide an additional means to tune transcription rates in a developmental context. Changes in RNA stability combine with changes in transcription to modulate mRNA pools during chloroplast development [78,80,81,84,85]. Determinants of chloroplast mRNA stability include various ribonucleases, RNA structure, ribosome occupancy, and proteins that protect RNAs from nuclease attack [8]. Most mRNA termini in chloroplasts are protected by helical repeat RNA binding proteins that provide a steric blockade to exoribonucleases [8,61,62,86]. The majority of such proteins belong to the pentatricopeptide repeat (PPR) family, a large family of sequence-specific RNA binding proteins that influence virtually every post-transcriptional step in gene expression in mitochondria and chloroplasts [87]. In addition, chloroplasts harbor abundant hnRNP-like proteins, and these have been shown to impact the stability of several chloroplast mRNAs [88,89]. Programmed changes in the abundance and/or activities of PPR and hnRNP-like proteins might contribute to the shifting mRNA pools during the proplastid to chloroplast transition. Changes in translational efficiency superimpose on changes in mRNA abundance to modulate the output of plastid genes during the transformation of proplastids into chloroplasts. ORFs encoding proteins involved in photosynthesis generally exhibit maximal translational efficiency in young or mature chloroplasts (segments 9 and 14), whereas those that function in gene expression generally peak in translational efficiency earlier in development (Fig 5C right). Furthermore, our data suggest that mRNAs encoding PSII reaction center proteins are translated with higher efficiency in mesophyll chloroplasts than in bundle sheath chloroplasts (Fig 6C). It will be interesting to explore the mechanisms that underlie these differential effects on translational efficiency. Some possibilities include shifts in stromal pH, Mg++, or the polymerase generating the mRNA (NEP versus PEP), which might impact the formation of RNA structures at specific ribosome binding sites. Programmed changes in the activities of nucleus-encoded gene-specific translational activators could modulate translational efficiencies in a developmental context. Most such proteins in land plant chloroplasts are PPR (or PPR-like) proteins, and several of these also stabilize processed mRNAs with a 5’ end at the 5’ boundary of their binding site [15,30,60–62,64–67,90–93]. Indeed, many polycistronic transcripts in chloroplasts are processed to smaller isoforms whose ends are defined and stabilized by PPR-like proteins [7,8, 86]. The impact of this type of RNA processing on translational efficiencies in vivo remains unclear. The removal of upstream ORFs is not required for the translation of several ORFs that are found on processed RNAs with a proximal 5’-terminus [35,36]. Some proteins have dual translation activation and RNA processing/stabilization functions, implying that the two activities are coupled [15,30,60–62,65,66,91–93]; however, the translation activation and RNA processing/ stabilization effects of such proteins could be independent consequences of their binding upstream of an ORF [62,86]. We showed here that there is a correlation between the accumulation of processed RNA isoforms and changes in relative translational efficiencies in two polycistronic transcription units (S8 Fig). Deciphering the cause and effect relationships underling these correlations presents a challenge for the future. The data presented here lead to numerous new questions for future exploration. Is the synthesis of nucleus-encoded subunits of photosynthetic complexes tuned to that of their chloroplast-encoded partners? What is the mechanistic basis for the preferential translation of some mRNAs in developing chloroplasts and others in photosynthetic chloroplasts? To what extent do environmental inputs such as light and temperature modify the developmental dynamics of chloroplast mRNA abundance and translation? The use of ribosome profiling can be anticipated to accelerate progress in addressing these and many other long-standing questions relating to the biology of organelles. For the developmental analysis, Zea mays (inbred line B73) was grown under diurnal cycles for 9 days and harvested as described [12]. Leaf sections from twelve plants were pooled for each of three replicates; each pool contained between 0. 15 g and 0. 3 g tissue. Plants used to prepare mesophyll and bundle sheath fractions were grown similarly, except the light was set at 300 μmol·m-2·s-1 and the tissues were harvested 13 days after planting, 2 hours into the light cycle. The apical one-third of leaf two and three were pooled from fifteen seedlings for each replicate, and the bundle sheath and mesophyll-enriched fractions were obtained with a rapid mechanical procedure. The tissue was cut into ~ 1 cm-sections, placed in a pre-chilled mortar and pestle, and lightly ground for 2 min in 5 ml of ice-cold modified polysome extraction buffer lacking detergents (0. 2 M sucrose, 0. 2 M KCl, 50 mM Tris-acetate, pH 8. 0,15 mM MgCl2,20 mM 2-mercaptoethanol, 2 μg/ml pepstatin A, 2 μg/ml leupeptin, 2 mM phenylmethanesulfonyl fluoride, 100 μg/ml chloramphenicol, 100 μg/ml cycloheximide). The material that was released into solution constituted the mesophyll cell-enriched fraction. A portion of this was frozen in liquid N2 for RNA isolation, and the remainder was stored on ice while bundle sheath strands were purified from the tissue remaining in the mortar. The tissue was subject to four additional rounds of light grinding (2 min), each time in a fresh aliquot of 5-ml modified polysome extraction buffer. The light green fibers remaining constituted the bundle sheath enriched fraction; these cells were broken by hard grinding in 5 ml of modified polysome extraction buffer. A portion of this material was flash frozen for future RNA isolation and the remainder was used immediately for ribosome footprint isolation. Polyoxyethylene (10) tridecyl ether and Triton X-100 were added to the mesophyll and bundle sheath fractions retained for ribosome profiling (final concentrations of 2% and 1%, respectively), and the material was filtered through glass wool. The isolation of ribosome footprints and total RNA were performed as described below. Ribosome footprints were prepared using a protocol similar to that described in [15], but with two key modifications: (i) RNAse I rather than micrococcal nuclease was used to generate monosomes, and (ii) the centrifugation time used to pellet ribosomes through the sucrose cushion was shortened to reduce contamination by other RNPs. Tissues were pulverized in liquid N2 with a mortar and pestle, and thawed in 5 ml of polysome extraction buffer (0. 2 M sucrose, 0. 2 M KCl, 50 mM Tris-acetate, pH 8. 0,15 mM MgCl2,20 mM 2-mercaptoethanol, 2% polyoxyethylene (10) tridecyl ether, 1% Triton X-100,100 μg/ml chloramphenicol, 100 μg/ml cycloheximide). A 2. 4-ml aliquot was removed and frozen in liquid N2 for total RNA isolation. The remaining suspension was filtered through glass wool and centrifuged at 15,000xg for 10 min. The supernatant was digested with 3,500 units of RNAse I (Ambion) at 23°C for 30 min. 2. 5 ml lysate was layered on a 2 ml sucrose cushion (1 M sucrose, 0. 1 M KCl, 40 mM Tris-acetate, pH 8. 0,15 mM MgCl2,10 mM 2-mercaptoethanol, 100 μg/ml chloramphenicol, and 100 μg/ml cycloheximide) in a 16 x 76 mm tube and centrifuged in a Type 80 Ti rotor for 1. 5 h at 55,000 rpm. The pellet was dissolved in 0. 7 mL of ribosome dissociation buffer (10 mM Tris-Cl, pH 8. 0,10 mM EDTA, 5 mM EGTA, 100 mM NaCl, 1% SDS). RNA was isolated with Tri reagent (Molecular Research Center). RNAs between ~20 and ~35 nt were purified on a denaturing polyacrylamide gel, eluted, extracted with phenol/chloroform, precipitated with ethanol, and suspended in water. We have subsequently modified our protocol to purify RNAs between 20 and 40 nt; this results in a small shift in the size distribution of the reads (S1B Fig). The ribosome footprint preparation was treated with T4 polynucleotide kinase. Twenty ng of the kinased RNA was converted to a sequencing library using the NEXTflex Small RNA Sequencing Kit v2 (Bioo Scientific), which minimizes ligation bias by introducing four randomized bases at the 3’ ends of the adapters [17]. rRNA fragments were depleted by subtractive hybridization after first-strand cDNA synthesis, using 54 biotinylated DNA oligonucleotides corresponding to the most abundant rRNA fragments detected in pilot experiments (see S1 Table). 10 μl of the oligonucleotide mixture (concentrations as in S1 Table) was added to 40-μl of the first-strand synthesis reaction and heated to 95°C for 2 min. A 50-μl aliquot of pre-warmed 2X hybridization buffer (10 mM Tris-Cl pH 7. 5,1 mM EDTA, 2 M NaCl) was added and incubated at 55°C for 30 min. The solution was transferred to a new tube containing 1 mg of prewashed Dynabeads M-270 Streptavidin (Invitrogen) and incubated at room temperature for 15 min with frequent agitation. The tube was placed on a magnet for 5 min and the supernatant was collected and desalted using Sephadex G-25 Fine (GE Healthcare). The sample was concentrated to 18 μl and used as input for the PCR amplification step in the library construction protocol. After 14 cycles, PCR products were separated by electrophoresis through a 5% polyacrylamide gel and a gel slice corresponding to DNA fragments between markers at 147 and 180 bp (representing insert sizes of 20–53 bp) was excised. The DNA was eluted overnight, phenol/chloroform extracted, precipitated with ethanol, suspended in water, and stored at -20°C. For RNA-seq, rRNA was depleted from the RNA samples using the Ribo-Zero rRNA Removal Kit (Plant Leaf) (Epicentre). One hundred ng of the rRNA-depleted RNA was used for library construction using the NEXTflex Rapid Directional qRNA-Seq Kit (Bioo Scientific) according the manufacturer’s instructions. The adapters provided with the kit include 8-nt molecular labels that were used during data processing to remove PCR bias. The libraries were combined and sequenced using a HiSeq 2500 or NextSeq 500 instrument (Illumina). The read lengths were 50 or 75 nt for Ribo-seq and 75 nt for mRNA-seq. Adapter sequences were trimmed using cutadapt [94]. Ribo-seq reads between 18 and 40 nt were used as input for alignments. Alignments were performed using Bowtie 2 with default parameters [95], which permits up to 2 mismatches, thereby allowing edited sequences to align. Reads were aligned to the following gene sets, with unaligned reads from each step used as input for the next round of alignment: (i) chloroplast tRNA and rRNA; (ii) chloroplast genome; (iii) mitochondrial tRNA and rRNA; (iv) mitochondrial genome (B73 AGP v3); (v) nuclear tRNA and rRNA; nuclear genome (B73 AGP v3). Maize genome annotation 6a (phytozome. jgi. doe. gov) was reduced to the gene set annotated in 5b+ (60,211 transcripts) (gramene. org). For metagene analysis, all coding sequence (CDS) coordinates from all transcript variants were combined to make a union CDS coordinate. Custom Perl scripts extracted mapping information using SAMtools [96] and analyzed mapped reads as follows. The distribution of ribosome footprint lengths and the RPKM for both the Ribo-seq and RNA-seq data were calculated based only on reads mapping to CDS regions. For RPKM calculations, we defined the total number of mapped reads as the number of reads mapping to nuclear CDSs. Translation efficiency was calculated from the division of ribosome footprint RPKM by RNA-seq RPKM. Because unspliced RNAs constitute a substantial fraction of the RNA pool from intron-containing genes in chloroplasts, these genes require special treatment to infer the abundance of spliced (functional) mRNA. The fraction spliced at each intron was calculated in several ways. (i) RNA-seq reads were aligned to the chloroplast genome with splicing-aware software TopHat 2. 0. 11 [97]. The number of reads spanning each exon-exon junction (spliced) was divided by the sum of spliced (exon-exon) and unspliced (exon 1-intron or intron-exon 2) reads; (ii) RNA-seq reads were aligned with Bowtie2 to a reference gene set that included both spliced and unspliced forms (100-nt on each side of each junction). The fraction of spliced RNA was calculated as for method (i); (iii) RNA-seq reads were aligned with TopHat to the genome and the spliced fraction was calculated from (exon RPKM—intron RPKM) /exon RPKM. Values calculated by each method are provided in S3 Table. Summary plots report mRNA abundance and translational efficiencies only for genes for which all of these methods gave similar results. We cannot confidently infer the amount of fully spliced RNAs from genes with two introns (ycf3 and rps12), so these are also excluded. Hierarchical clustering was performed using the Bioinformatic Toolbox of MATLAB software (Mathworks) using standardized values as input: values from the four leaf segments for each gene were standardized to have a mean of 0 and a standard deviation of 1 such that developmental shifts can be compared among genes despite differences in signal magnitude. Hierarchical clustering was performed using Pearson correlation coefficient values and unweighted average distance. Antibodies to AtpB, D1 and PetD were raised by our group and have been described previously [30]. Antibodies to Atp6, NdhH, PPDK, and Rpl2 were generously provided by Christine Chase (University of Florida), Tsuyoshi Endo (Kyoto University), Kazuko Aoyagi (UC Berkeley), and Alap Subramanian (University of Arizona), respectively. Antibodies to PEPC, malic enzyme, and RbcL were generous gifts of William Taylor (University of California, Berkeley). Illumina read sequences were deposited at the NCBI Sequence Read Archive with accession number SRP070787. Alignments of reads to the maize chloroplast genome used Genbank accession X86563. | Title: Dynamics of Chloroplast Translation during Chloroplast Differentiation in Maize Summary: Chloroplasts are subcellular organelles in plants and algae that carry out the core reactions of photosynthesis. Chloroplasts originated as cyanobacterial endosymbionts. Subsequent coevolution with their eukaryotic host resulted in a massive transfer of genes to the nuclear genome, the acquisition of new gene expression mechanisms, and the integration of chloroplast functions into host programs. Chloroplasts in multicellular plants develop from non-photosynthetic proplastids, a process that involves a prodigious increase in the expression of chloroplast genes encoding components of the photosynthetic apparatus. We used RNA sequencing and ribosome profiling to generate a comprehensive description of the dynamics of chloroplast gene expression during the transformation of proplastids into the distinct chloroplast types found in bundle sheath and mesophyll cells in maize. Genes encoding proteins that make up the chloroplast gene expression machinery peak in protein output earlier in development than do those encoding proteins that function in photosynthesis. Programmed changes in translational efficiencies superimpose on changes in mRNA abundance to shift the balance of protein output as chloroplast development proceeds. We also mined the data to gain insight into general features of chloroplast gene expression, such as relative translational efficiencies, the impact of RNA editing on translation, and the identification of rate limiting steps in gene expression. The findings clarify the parameters that dictate the abundance of chloroplast gene products and revealed unanticipated phenomena to be addressed in future studies. | 13,241 | 349 | lay_plos | en |
Summarize: Two men are halfway up what has been called the hardest rock climb in the world - a free climb up a half-mile section of exposed granite in California's Yosemite National Park. Tommy Caldwell, 36, of Estes Park, Colorado, and Keven Jorgeson, 30, of Santa Rosa, California, have been scaling their way up the face of the 3,000 feet granite monolith of El Capitan in Yosemite using only their hands and feet. Most ascents up the imposing rock face use an approach known as aid climbing, where ropes and metal pins are used to assist the climbers. Don't look down: Jorgeson calculates his next move up the mountain in this photo posted on Instagram on Sunday. However, the Caldwell and Jorgeson are instead attempting to cleanly free climb a route that has never before been climbed without aid. Their progress up the 32 sections, or pitches, of climbing is being chronicled by climber and photographer Tom Evans and filmmaker Brett Lowell. The two men eat, stretch and sleep in hanging tents suspended to El Capitan's Dawn Wall. They don't have the creature comforts of home, but they have kept in touch with the outside world thanks to social media - tweeting, posting on Facebook, feeding information for blogs and keeping in touch with a bevy of supporters on the ground. Time to relax: On Monday, the pair took a rest day to heal their hands in preparation for another day on the rock face on Tuesday. Above, Kevin Jorgeson relaxes in his portaledge tent, which is attached to the rock face and hangs above the Yosemite Valley floor far below. Daring: Free climbing involves using ropes to protect the men should they fall while climbing but it does not actually aid their ascent. Above, Caldwell makes his way up one of the thin cracks that work their way up the route on El Capitan using a headlamp on Sunday night. Up they go: Kevin Jorgeson, 30 (left), and Tommy Caldwell, 36 (right), have reached the halfway point on their attempt to free climb Yosemite's El Capitan. Record breakers: If they are successful, Jorgeson and Caldwell will be the first team to free climb El Capitan's imposing Dawn Wall. Rock face life: On the left, Caldwell is seen on Day 9 of the climb, attempting to remove damaged skin on his hands. The razor-sharp rock on the wall tears up the climber's fingertips and they use sanding blocks to keep them smooth. On the right, one of the meals the climbers have eaten on El Capitan. The pair keep fueled with sandwiches like this and in the cold conditions their food is practically refrigerated. 'The guys are doing great,' said Josh Lowell with Big Up Productions, which has been filming the pair as they have battled with the route over the past six years. '(Monday) they are resting and trying to grow skin back on their fingertips so they can continue to do battle with the hardest climbing sections, which involve grabbing tiny, razor-sharp edges of rock.' If all goes as planned, the duo could be at the top as soon as Friday or Saturday, Lowell said. 'But that's best-case scenario. It could take several more days just to get through the difficult section where they currently are. If any weather moves in, that could also delay things, but the forecast is looking good for now,' he said. It will be the culmination of a six year project to conquer the route that they have called the Dawn Wall. Until now the pair have been gradually edging their way up the route one pitch at a time, practicing each move until they have been able to climb freely up the holds without falling or the need for assistance from aid climbing equipment. Previous attempts to climb the route cleanly have been beset with problems. Caldwell and Jorgeson tried to climb it all in 2010, but storms halted their progress about a third of the way up while in 2011 Jorgeson broke an ankle after falling during an attempt. Just after Christmas 2014 the pair began their latest attempt to finally climb the entire route without stopping. Progress: If all goes well, the pair could reach the summit of El Capitan as early as Friday or Saturday. Above, Caldwell makes his way up the rock face on Sunday. Daunting: At 3,000-feet-tall, El Capitan is the largest monolith of granite in the world and one of the most difficult climbs. Many have climbed Dawn Wall but the pair would be the first to 'free climb' the section using ropes only as a safeguard against falls. Previous ascensionist have used ropes and metal pitons to help pull themselves upwards, as well as to stop them plummeting to the ground if they fall. Caldwell and Jorgenson are pulling themselves up the route using only the tiny holds they can find on the rock. For Caldwell this has meant overcoming a DIY accident where he cut off much of his left index finger with a table saw in 2001. He has trained with his remaining figures to build up their strength. But the razor sharp granite holds on Dawn Wall has now led both Caldwell and Jorgeson to tape their finger tips as the rock has shredded their skin. The first climber reached El Capitan's summit in 1958, and there are roughly 100 routes up to the top. Evans said the two have a cellphone on their ascent, but they weren not taking calls Monday because they were resting and 'want no distractions while on the cliff.' The two also were not answering emails from roughly 1,500 feet above the ground. Not out of the loop: The pair keep in touch with the outside world using a cellphone they brought with them on the climb, using it to tweet and post updates to the social networking site Facebook. However, on Monday they stayed off the device to focus on resting. These practices may not seem unusual, but the climbers have relied heavily on social media to document their adventure. Both update their Facebook pages regularly and tweet from the Dawn Wall, which has been called 'as smooth as alabaster, as steep as the bedroom wall.' Last Friday, Jorgeson hosted a live question-and-answer session from the wall. Mountaineers: The first climber reached El Capitan's summit in 1958, and there are roughly 100 routes up the steep rock faces to the top. Live from El Capitan: On Friday, Jorgeson hosted a question and answer session from the wall. Seen climbing El Capitan, above. Worst is over: There are 32 sections of El Capitan to climb, but the pair have already finished the three hardest sections of the route. Caldwell's wife', Becca, has also been blogging about their trip daily and wrote this post last weekend. 'Being up on the wall for over a week and the hard climbing Tommy and Kevin have done up until now adds an element of difficulty on top of the hard climbing they have to do,' she wrote. 'Imagine performing your very best after not walking for one week. I know Tommy has made an effort to try and do stretching, pushups, (and) yoga in the (hanging tent) hoping this might combat the unusual circumstances of living like veal between their climbing. So let's hope for big things today. This climb definitely won't be over until it's over, but I believe it's possible. Let's go boys!!!' Jorgeson tweeted late Saturday about his difficulty scaling one section: 'Battling. #dawnwall.' On December 30 the pair managed to compete the hardest section of the wall - the first traverse at around pitch 14. Writing on Facebook later that evening, Jorgeson said: 'Tonight, I sent the hardest pitch of my life and the hardest on the Dawn Wall! Best of all, Tommy sent right after me! Pitch 14 (the first traverse) is in the bag!' Caldwell added: 'The hardest pitch got sent by both of us tonight. I might be in a little shock right now. The route is taking a toll on our fingertips as we are now both climbing with taped up fingertips, but it doesn't seem to be slowing us down too much.' There are 32 sections of the climb. On Sunday night, Lowell said Caldwell, climbing in the dark, completed the last of the three hardest sections of climbing, which was a major breakthrough, Lowell said. 'He still has 1,500 feet of hard, scary climbing ahead, but mentally he is feeling really confident right now, and incredibly excited. (Jorgeson) is extremely close to completing pitch 15, one of the hardest. (Tuesday) he will try to complete it and catch up to Tommy so they can continue forging ahead.' In 1970, Warren Harding and Dean Caldwell (no relation to Tommy Caldwell) climbed Dawn Wall using ropes and countless rivets over 27 days. The duo prepared for at least six years for the climb, according to friends and their personal websites. John Long, the first person to climb El Capitan in one day in 1975, said he speaks to the climbers several times a day. 'It's almost inconceivable that anyone could do something that continuously difficult,' he said Monday, adding that he believes they spent the equivalent of a year's time on the wall in preparation for the climb. To follow Jorgeson and Caldwell on their journey, follow them on Instagram. Big UP Productions and photographer Corey Rich are also capturing the two as they ascend the monolith, and both have been posting pictures of the monumental climb on Instagram as well | Summary: Climbers Kevin Jorgeson, 30, and Tommy Caldwell, 36, are making their way up El Capitan - the largest monolith of granite in the world - in a single push without pulling on any equipment to aid their ascent. Many have climbed El Capitan before, but if successful, the pair would be the first to 'free climb' a route known as the Dawn Wall in what is considered to be the most difficult multipitch climb in the world. Caldwell is doing the climb despite cutting off much of the index finger of his left hand in a DIY accident with a saw. Razor sharp holds on the rock have taken their toll on the pair and they are now climbing with their fingertips taped. Free climbing involves using ropes and clips to protect in case of a fall, but does not aid the actual ascent. Jorgeson and Caldwell hope to reach the summit of El Capitan as by Friday or Saturday to end a six year project. | 2,187 | 207 | cnn_dailymail | en |
Summarize: NEWS StudCo passes resolution addressing Boys’ Bid Night ban Seeks national sorority discussions Student Council passed a resolution Tuesday addressing the self-governance of sorority women, urging national sorority organizations to respect University principles of student self-governance as well as University-approved sorority safety plans. The resolution follows a letter from the National Panhellenic Conference on behalf of 16 national and international sorority presidents requesting University chapters not participate in organized Boys’ Bid Night activities. Several sorority chapters have subsequently confirmed their national chapters are requiring individual sorority members to not participate in Bid Night activities, with several mandating participation in alternative chapter events that evening. Engineering Rep. Danielle Ager, a fourth year and co-sponsor of the resolution, said it was important to protect student self governance and encourage collaboration between national sorority organizations and University chapters. “What [is] really an issue is there’s no respect given with how we operate at U.Va. with student self-governance,” she said. “[The mandate] just appeared one day and everyone was expected to comply. The issue is not that we can’t go out on Boys’ Bid Night but what precedent this is setting for the future.” Third-year Commerce student Faith Lyons, Council director of University relations, said while it is possible a collaborative solution to sorority safety concerns may have still included a decision to not participate in Boys’ Bid Night, the University community should have been part of the decision-making process. “Student self-governance is part of everything we do on Grounds,” she said. “Last semester students were a part of the conversation and part of creating the solution.” Safety and Wellness Chair Rachel Murphy, a third-year College student, said the NPC mandate actually reduces the overall safety of students and brings into question female agency at the University. “Sorority women are being used as a pawn for fraternity men to get their act together,” she said. “You miss out on all these women who are trained to be active bystanders.” Murphy also said in prohibiting sorority participation in Boys’ Bid Night, fraternities lose the opportunity to show the University their improved safety standards. Ager said the mandate would overshadow the recently adopted University-approved safety measures. “There was a safety plan made at the end of last semester that all the fraternities and sororities signed at the beginning of this semester,” she said. “This gets in the way of chapters trying to implement that.” Inter-Sorority Council President Allison Palacios, a third-year College student, said national organizations are implementing the mandate in an effort to promote safety among their members. “From my conversation with leaders of the organization this past weekend, they are keeping in mind women’s leadership,” she said. “At the same time they are trying to act in the way they know best at this point.” Palacios said there are also other important safety considerations — like binge drinking and physical injuries — which have fallen out of the conversation. “I think that national organizations have been naïve to not address this situation in the past,” she said. “I understand that there should’ve been a conversation, but at the same time we had a conversation that upon entering a sorority you’re always wearing your letters and are held up to a certain standard. Additionally, I’d like to point out that this mandate is not supposed to be new.... Women are not supposed to be involved in men’s recruitment.” Fourth-year College student Olivia Bona took issue with the apparent unwillingness of national organizations to consider the complexities and conflicting sides of the issue, such as an individual’s ability to act on their own behalf and not as a member of a sorority. “Yes, I chose to agree to be in a sorority,” she said. “However, as an individual I should be able to do whatsoever I choose.” Ager said it is important to stress the importance of collaboration between national organizations and students. “The main thing Student Council is asking for is for the national organizations to engage with their members,” she said. Council invited the president of each National Sorority Organization to Charlottesville for further discussion the issue Friday. The University of Virginia campus. (Rebecca Drobis/ For The Washington Post) Sorority sisters at the University of Virginia were ordered by their national chapters to avoid fraternity events this weekend — a mandate that many of the women said was irrational, sexist and contrary to the school’s culture. It’s not about one night of parties, several students said, but about their ability to make their own choices. And they’re not taking that lightly. The rule came after a traumatic fall semester in Charlottesville, including the violent death of a student and now-discredited allegations of a gang rape at a U-Va. fraternity. Both forced a thorough examination of campus safety, drinking culture and Greek life. WUVA online, a student-run media outlet, interviewed Nicole Eramo, an associate Dean of Students who heads the university's Sexual Misconduct Board. According to WUVA, the interview took place before a Rolling Stone story about an alleged gang rape at a fraternity at the University of Virginia. WUVA published the interview on Saturday. (WUVA INC.) The university administration just days ago lifted a suspension of fraternity and sorority activities that came in the wake of the sexual assault allegations, a break that the university community used to have a broad discussion about student safety in the Greek system. In order to have the ban lifted — immediately ahead of spring rush, when students join fraternities — the school’s Greek houses agreed to new rules amid new student-led initiatives to increase safety. Many students had been looking forward to celebrating with old friends and new members during the fraternities’ bid night, scheduled for Saturday, Jan. 31. There are 16 sororities on U-Va.’s campus that are part of the National Panhellenic Conference, with more than 2,000 members, according to the campus Inter-Sorority Council. The NPC can come to “unanimous agreements” among its national presidents that are binding on local chapters and their members. At some U-Va. chapters in recent days, students described mandatory emergency meetings with representatives from their national chapter telling them they risked suspension, fines and other penalties if any of them attended bid night parties. Boys’ Bid Night is typically a night when sorority sisters go from house to house sharing drinks with friends. Now some sororities are planning mandatory in-house retreats that night, to avoid any risk of inadvertently violating the rule. At some chapters, women were told not only to avoid going to fraternity parties on Boys’ Bid Night, but to avoid any social gathering with fraternity members, said Ben Gorman, president of the Inter-Fraternity Council at U-Va. That would mean a ban on attending off-campus parties or gatherings at bars that night after a hotly anticipated basketball game on campus, which pits the undefeated No. 2 Cavaliers against No. 4 Duke. “People are very agitated and very upset, and see this as an obstacle to larger cultural change a violation of free rights and student free will.” A university spokesman deferred questions to the National Panhellenic Conference, as did the incoming president of the Inter-Sorority Council at U-Va. A spokeswoman for the National Panhellenic Conference said the mandate comes not from the umbrella group but from each national chapter president. “Of course, NPC supports the safety of their women, so they do support those national presidents making that decision and encouraging sorority women to plan sisterhood events and other ‘safer’ options,” Michelle Bower said. An emergency Student Council bill aimed at addressing the issue passed 14-0-3 on Tuesday night, urging the national chapter leaders to join students on campus to talk about the issue Friday. “This was entirely top-down — an edict,” said Abraham Axler, a second-year student who is chair of the representative body. “That is not how things operate at U-Va.” A petition, started online on Monday, had almost 2,000 signatures by midday Tuesday. It reads, in part: “Instead of addressing rape and sexual assault at UVa, this mandate perpetuates the idea that women are inferior, sexual objects. It is degrading to Greek women, as it appears that the [National Panhellenic Conference] views us as defenseless and UVa’s new fraternal policies as invalid. Allowing the NPC to prevent us from celebrating (what used to be) a tight-knit community, sends the message that we are weak.” And another widely-circulated letter, directed to the National Panhellenic Conference, reads, in part: “Our concerns lie in the way sorority women are being used as leverage to change the actions and behaviors of fraternity men. This resolution has misconstrued us as a passive aggregate rather than active agents for change. It has also had the unintended consequence of subjugating women. … Women have historically been the targets of sexual violence, and forbidding us to exercise our agency plays dangerously into gender stereotypes surrounding the issue.” The mandate is diametrically opposed to the central values of the sororities in fostering and supporting women’s strength, the letter continues, and, “This solution is not long-term, realistic, or sustainable.” | Summary: Sorority members at the University of Virginia are taking a stand over a rule they argue is rooted in discrimination. This Saturday is bid night at UVa campus fraternities, an event in which the sorority sisters normally take part-but this year, national chapters are requiring many of them to avoid the parties, the Washington Post reports. The national chapters' decision follows the death of a student as well as allegations of gang rape at a fraternity house. Though those allegations were later said to be unsubstantiated, the university has taken a hard look at safety on campus, the Post notes. Now, some women are even banned from participating in events with fraternity members off-campus that night. Says the National Panhellenic Organization that oversees Greek life: "NPC supports the safety of their women, so they do support those national presidents making that decision and encouraging sorority women to plan sisterhood events and other'safer' options." A letter to the NPC gives voice to those opposed to the policy: "Sorority women are being used as leverage to change the actions and behaviors of fraternity men," it reads. "This resolution has misconstrued us as a passive aggregate rather than active agents for change." Meanwhile, an online petition to revoke the ban has gathered more than 2,000 signatures. The school's Student Council yesterday passed a resolution calling on the national groups to change the mandate, the Cavalier Daily reports. | 2,082 | 317 | multi_news | en |
Summarize: By. Daily Mail Reporter. PUBLISHED:. 18:35 EST, 27 February 2014. |. UPDATED:. 01:56 EST, 28 February 2014. New York Knicks point guard Raymond Felton not only has run afoul of the law over weapons possession, but he also landed in trouble with his wife over allegations of constant infidelities. The ball player was arraigned on two felony weapons possession charges in Manhattan Criminal Court Tuesday and then released on $25,000 bail following an early morning weapons possession arrest. Earlier Felton turned himself in to authorities after a lawyer for his estranged wife of only 19 months turned in a loaded semi-automatic handgun allegedly belonging to the Knicks star to a police precinct, claiming she no longer wanted it in their home. Scroll down for video. Scorned wife: Ariane Raymondo-Felton (left), 24, told police her husband, Knicks star Raymond Felton (right), had multiple mistresses and had allegedly threatened her with a gun during domestic squabbles. Not sweating it? New York Knicks point guard Raymond Felton appeared largely upbeat at his Manhattan arraignment Tuesday alongside one of his attorneys. Law enforcement sources told the New. York Post that Ariane Raymondo-Felton, the 24-year-old brunette beauty. who walked down the aisle with the athlete in 2012, told police that. Felton had been juggling several paramours on the side. ‘He's an NBA player. He flirts and stuff,’ a source said. ‘When you're in a position like that you could get 10 girls per week.’ Raymondo-Felton,. who is a student at the prestigious Fordham Law School, also told. police that she and her philandering husband had four fights since the. summer, and on two occasions Felton pulled his FNH Five-Seven 28mm. handgun on her. The final altercation between the newlyweds came on Valentine's Day, which erupted over domestic problems. During. the fight, Felton’s wife claimed that the 29-year-old star athlete. brandished the gun in front of her to 'intimidate' her, the woman later. told police. Raymondo-Felton filed for divorce four days after the confrontation with her husband. The. law student's attorney advised her to turn over her husband's. unregistered handgun, which Felton had been keeping under their bed in. the couple's apartment on West 63rd Street. Rumors. of Felton’s extramarital trysts have long been swirling around the. dashing Knicks player - and they have even made it into a song. Rapper Fabolous allegedly alluded to Felton's status as an avowed ladies' man in the ‘Cuffin Season' lyrics. ‘4. Celtics, Knicks guard, you know Felton,’ he rapped. ‘This n—- in. Atlanta AirTran her, no Delta. Been naughty all year trying to end it. nicely, Summer hoes turning into winter wifeys.’ Ladies' man: It has been rumored that Felton, 29, had multiple affairs since tying the knot in July 2012. In their wedding video from July 2012, Felton vowed to be true to his leading lady. ‘Beautiful. as ever, from the first day I met her,’ the Knicks player declared. ‘I’m going to take care of her. You have my word. I promise you. No. matter what it is, I’m always there for you. I love you.’ Wearing. a black sweatshirt with a peace sign and other symbols on it, Felton. was seemingly upbeat as he appeared before Judge Diana Boyar, nodding. affirmatively after he was ordered to stay away from his wife, Ariane. Raymondo-Felton. He did not enter a plea, which is common for this stage. in the case. 'Mr. Felton has no interest in having. contact' with her, one of his lawyers, James Walden, told the judge. Court records show she filed for divorce from Felton last week. Felton. was released on $25,000 bail and was ushered into a black SUV following. his arraignment. Under the terms of his bond, Felton can travel to. games, bail bondsman Ira Judelson said. Prosecutors. said they were told Felton stored the Belgian-made FN Herstal model. handgun in the home from August through February. A lawyer for Felton's. wife, a Cornell University graduate, dropped off the. weapon at a station house on Manhattan's upper West Side Monday. evening, shortly before tipoff of the Knicks game against the Dallas. Mavericks at Madison Square Garden, police said. Wife: A lawyer for Felton's wife, a student at Fordham University School of Law, dropped off the weapon at a stationhouse on Manhattan's upper West Side on Monday evening, shortly before tipoff of the Knicks game against the Dallas Mavericks at Madison Square Garden. Final straw: Raymondo-Felton filed for divorce just days after a Valentine's Day fight with her husband, during which the man allegedly waved his handgun in her face. The gun had 18 rounds of live ammunition in its magazine, which can hold about 20 rounds, prosecutors said. He was charged with criminal possession of a weapon in the third degree and criminal possession of a firearm. The firearm charge is punishable by up to four years in prison. The weapons charge is punishable by up to seven years in prison. The section under which he was charged concerns having a large-capacity ammunition magazine. Police had arrested Felton on charges that included a mid-level weapons-possession charge that can entail having a loaded gun outside one's home or business or having a loaded gun with the intention to use it against someone. The DA's office didn't comment on why prosecutors chose the charges they did; it's not uncommon for charges to change between arrest and arraignment. Investigators reached Felton by. contacting the director of security at Madison Square Garden after his. wife made a statement to detectives Monday night, police said. Felton. turned himself in at 12:50 a.m. Tuesday, not long after the Knicks' buzzer-beater loss to Dallas, police said. The former University of North Carolina star made no statement after he arrived at the precinct with a lawyer, police said. A. Knicks spokesman has said the team had no immediate comment. An. attorney for Raymondo-Felton didn't comment. The Knicks had no game. scheduled Tuesday. Released: Raymond Felton was arrested early Tuesday on felony weapons charges and is seen here leaving Manhattan Criminal Court after his arrangement and making $25,000 bail. Free to play: Felton was ushered into a black SUV following his arraignment. Under the terms of his bond, Felton can travel to games. Felton had eight points and seven assists Monday in the Knicks' 110-108 loss to Dallas. The Knicks brought the point guard back for a second stint in New York in July 2012, opting to let Jeremy Lin leave, and Felton helped the Knicks win the Atlantic Division last year. But he has had a disappointing season, averaging 10.4 points and shooting 40 percent while missing 16 games with a series of injuries. He has been frequently criticized by fans as the Knicks have fallen to 21-36. Felton had allegedly stored an FN Herstal similar to this in his Upper West Side apartment since April. NBA spokesman Tim Frank said the league was monitoring the case. It could fine or suspend Felton, but usually waits until after the legal case has been resolved. The league did break from that policy. to suspend Gilbert Arenas for the remainder of the season after he. brought guns to the Washington Wizards' locker room during the 2009-10. season. He was eventually sentenced to 30 days in a halfway house. Felton. is not the first pro athlete to run afoul of New York's strict gun. laws. Plaxico Burress was a New York Giants wide receiver when he was. arrested in 2008 after accidentally shooting himself in a Manhattan. nightclub. He served two years in prison. Fighter. Robert Guerrero was arrested at John F. Kennedy Airport last year after. he presented a locked gun box containing an unloaded handgun during. check-in. He pleaded guilty to disorderly conduct and was ordered to pay. a $250 fine and complete 50 hours of community service. In. other high-profile cases, multiplatinum-selling rappers Lil Wayne and. Ja Rule both were arrested in Manhattan, separately, on gun-possession. charges in their vehicles after leaving the same concert they had both. played in 2007. Both pleaded guilty to attempted gun possession charges;. Lil Wayne spent about eight months in a city jail; Ja Rule served most. of a two-year prison sentence. Felton. is set to earn $3.8million next season and has a player option that. would pay him $4million in 2015-16. The Knicks could attempt to void. the contract, but that would likely be challenged by the players' association | Summary: Felton, 29, turned himself in to police at 12:50am Tuesday. He was arrested on three counts of criminal possession of a weapon thanks to New York's strict gun laws. Felton appeared upbeat at his arraignment where attorneys said he has no desire to make contact with estranged wife Ariane Raymondo-Felton. Miss Raymondo-Felton, 24, allegedly told police her husband of 19 months had been cheating on her. Accused basketball star of trying to intimidate her with his handgun on at least two occasions. Rapper Fabolous sang about Felton's alleged infidelities in 'Cuffin Season' | 2,244 | 151 | cnn_dailymail | en |
Summarize: Her fit of weeping, however, was soon smothered, and the signs of it had vanished when, an hour later, she broke the news to her aunt. I use this expression because she had been sure Mrs. Touchett would not be pleased; Isabel had only waited to tell her till she had seen Mr. Goodwood. She had an odd impression that it would not be honourable to make the fact public before she should have heard what Mr. Goodwood would say about it. He had said rather less than she expected, and she now had a somewhat angry sense of having lost time. But she would lose no more; she waited till Mrs. Touchett came into the drawing-room before the mid-day breakfast, and then she began. "Aunt Lydia, I've something to tell you." Mrs. Touchett gave a little jump and looked at her almost fiercely. "You needn't tell me; I know what it is." "I don't know how you know." "The same way that I know when the window's open--by feeling a draught. You're going to marry that man." "What man do you mean?" Isabel enquired with great dignity. "Madame Merle's friend--Mr. Osmond." "I don't know why you call him Madame Merle's friend. Is that the principal thing he's known by?" "If he's not her friend he ought to be--after what she has done for him!" cried Mrs. Touchett. "I shouldn't have expected it of her; I'm disappointed." "If you mean that Madame Merle has had anything to do with my engagement you're greatly mistaken," Isabel declared with a sort of ardent coldness. "You mean that your attractions were sufficient, without the gentleman's having had to be lashed up? You're quite right. They're immense, your attractions, and he would never have presumed to think of you if she hadn't put him up to it. He has a very good opinion of himself, but he was not a man to take trouble. Madame Merle took the trouble for him." "He has taken a great deal for himself!" cried Isabel with a voluntary laugh. Mrs. Touchett gave a sharp nod. "I think he must, after all, to have made you like him so much." "I thought he even pleased YOU." "He did, at one time; and that's why I'm angry with him." "Be angry with me, not with him," said the girl. "Oh, I'm always angry with you; that's no satisfaction! Was it for this that you refused Lord Warburton?" "Please don't go back to that. Why shouldn't I like Mr. Osmond, since others have done so?" "Others, at their wildest moments, never wanted to marry him. There's nothing OF him," Mrs. Touchett explained. "Then he can't hurt me," said Isabel. "Do you think you're going to be happy? No one's happy, in such doings, you should know." "I shall set the fashion then. What does one marry for?" "What YOU will marry for, heaven only knows. People usually marry as they go into partnership--to set up a house. But in your partnership you'll bring everything." "Is it that Mr. Osmond isn't rich? Is that what you're talking about?" Isabel asked. "He has no money; he has no name; he has no importance. I value such things and I have the courage to say it; I think they're very precious. Many other people think the same, and they show it. But they give some other reason." Isabel hesitated a little. "I think I value everything that's valuable. I care very much for money, and that's why I wish Mr. Osmond to have a little." "Give it to him then; but marry some one else." "His name's good enough for me," the girl went on. "It's a very pretty name. Have I such a fine one myself?" "All the more reason you should improve on it. There are only a dozen American names. Do you marry him out of charity?" "It was my duty to tell you, Aunt Lydia, but I don't think it's my duty to explain to you. Even if it were I shouldn't be able. So please don't remonstrate; in talking about it you have me at a disadvantage. I can't talk about it." "I don't remonstrate, I simply answer you: I must give some sign of intelligence. I saw it coming, and I said nothing. I never meddle." "You never do, and I'm greatly obliged to you. You've been very considerate." "It was not considerate--it was convenient," said Mrs. Touchett. "But I shall talk to Madame Merle." "I don't see why you keep bringing her in. She has been a very good friend to me." "Possibly; but she has been a poor one to me." "What has she done to you?" "She has deceived me. She had as good as promised me to prevent your engagement." "She couldn't have prevented it." "She can do anything; that's what I've always liked her for. I knew she could play any part; but I understood that she played them one by one. I didn't understand that she would play two at the same time." "I don't know what part she may have played to you," Isabel said; "that's between yourselves. To me she has been honest and kind and devoted." "Devoted, of course; she wished you to marry her candidate. She told me she was watching you only in order to interpose." "She said that to please you," the girl answered; conscious, however, of the inadequacy of the explanation. "To please me by deceiving me? She knows me better. Am I pleased to-day?" "I don't think you're ever much pleased," Isabel was obliged to reply. "If Madame Merle knew you would learn the truth what had she to gain by insincerity?" "She gained time, as you see. While I waited for her to interfere you were marching away, and she was really beating the drum." "That's very well. But by your own admission you saw I was marching, and even if she had given the alarm you wouldn't have tried to stop me." "No, but some one else would." "Whom do you mean?" Isabel asked, looking very hard at her aunt. Mrs. Touchett's little bright eyes, active as they usually were, sustained her gaze rather than returned it. "Would you have listened to Ralph?" "Not if he had abused Mr. Osmond." "Ralph doesn't abuse people; you know that perfectly. He cares very much for you." "I know he does," said Isabel; "and I shall feel the value of it now, for he knows that whatever I do I do with reason." "He never believed you would do this. I told him you were capable of it, and he argued the other way." "He did it for the sake of argument," the girl smiled. "You don't accuse him of having deceived you; why should you accuse Madame Merle?" "He never pretended he'd prevent it." "I'm glad of that!" cried Isabel gaily. "I wish very much," she presently added, "that when he comes you'd tell him first of my engagement." "Of course I'll mention it," said Mrs. Touchett. "I shall say nothing more to you about it, but I give you notice I shall talk to others." "That's as you please. I only meant that it's rather better the announcement should come from you than from me." "I quite agree with you; it's much more proper!" And on this the aunt and the niece went to breakfast, where Mrs. Touchett, as good as her word, made no allusion to Gilbert Osmond. After an interval of silence, however, she asked her companion from whom she had received a visit an hour before. "From an old friend--an American gentleman," Isabel said with a colour in her cheek. "An American gentleman of course. It's only an American gentleman who calls at ten o'clock in the morning." "It was half-past ten; he was in a great hurry; he goes away this evening." "Couldn't he have come yesterday, at the usual time?" "He only arrived last night." "He spends but twenty-four hours in Florence?" Mrs. Touchett cried. "He's an American gentleman truly." "He is indeed," said Isabel, thinking with perverse admiration of what Caspar Goodwood had done for her. Two days afterward Ralph arrived; but though Isabel was sure that Mrs. Touchett had lost no time in imparting to him the great fact, he showed at first no open knowledge of it. Their prompted talk was naturally of his health; Isabel had many questions to ask about Corfu. She had been shocked by his appearance when he came into the room; she had forgotten how ill he looked. In spite of Corfu he looked very ill to-day, and she wondered if he were really worse or if she were simply disaccustomed to living with an invalid. Poor Ralph made no nearer approach to conventional beauty as he advanced in life, and the now apparently complete loss of his health had done little to mitigate the natural oddity of his person. Blighted and battered, but still responsive and still ironic, his face was like a lighted lantern patched with paper and unsteadily held; his thin whisker languished upon a lean cheek; the exorbitant curve of his nose defined itself more sharply. Lean he was altogether, lean and long and loose-jointed; an accidental cohesion of relaxed angles. His brown velvet jacket had become perennial; his hands had fixed themselves in his pockets; he shambled and stumbled and shuffled in a manner that denoted great physical helplessness. It was perhaps this whimsical gait that helped to mark his character more than ever as that of the humorous invalid--the invalid for whom even his own disabilities are part of the general joke. They might well indeed with Ralph have been the chief cause of the want of seriousness marking his view of a world in which the reason for his own continued presence was past finding out. Isabel had grown fond of his ugliness; his awkwardness had become dear to her. They had been sweetened by association; they struck her as the very terms on which it had been given him to be charming. He was so charming that her sense of his being ill had hitherto had a sort of comfort in it; the state of his health had seemed not a limitation, but a kind of intellectual advantage; it absolved him from all professional and official emotions and left him the luxury of being exclusively personal. The personality so resulting was delightful; he had remained proof against the staleness of disease; he had had to consent to be deplorably ill, yet had somehow escaped being formally sick. Such had been the girl's impression of her cousin; and when she had pitied him it was only on reflection. As she reflected a good deal she had allowed him a certain amount of compassion; but she always had a dread of wasting that essence--a precious article, worth more to the giver than to any one else. Now, however, it took no great sensibility to feel that poor Ralph's tenure of life was less elastic than it should be. He was a bright, free, generous spirit, he had all the illumination of wisdom and none of its pedantry, and yet he was distressfully dying. Isabel noted afresh that life was certainly hard for some people, and she felt a delicate glow of shame as she thought how easy it now promised to become for herself. She was prepared to learn that Ralph was not pleased with her engagement; but she was not prepared, in spite of her affection for him, to let this fact spoil the situation. She was not even prepared, or so she thought, to resent his want of sympathy; for it would be his privilege--it would be indeed his natural line--to find fault with any step she might take toward marriage. One's cousin always pretended to hate one's husband; that was traditional, classical; it was a part of one's cousin's always pretending to adore one. Ralph was nothing if not critical; and though she would certainly, other things being equal, have been as glad to marry to please him as to please any one, it would be absurd to regard as important that her choice should square with his views. What were his views after all? He had pretended to believe she had better have married Lord Warburton; but this was only because she had refused that excellent man. If she had accepted him Ralph would certainly have taken another tone; he always took the opposite. You could criticise any marriage; it was the essence of a marriage to be open to criticism. How well she herself, should she only give her mind to it, might criticise this union of her own! She had other employment, however, and Ralph was welcome to relieve her of the care. Isabel was prepared to be most patient and most indulgent. He must have seen that, and this made it the more odd he should say nothing. After three days had elapsed without his speaking our young woman wearied of waiting; dislike it as he would, he might at least go through the form. We, who know more about poor Ralph than his cousin, may easily believe that during the hours that followed his arrival at Palazzo Crescentini he had privately gone through many forms. His mother had literally greeted him with the great news, which had been even more sensibly chilling than Mrs. Touchett's maternal kiss. Ralph was shocked and humiliated; his calculations had been false and the person in the world in whom he was most interested was lost. He drifted about the house like a rudderless vessel in a rocky stream, or sat in the garden of the palace on a great cane chair, his long legs extended, his head thrown back and his hat pulled over his eyes. He felt cold about the heart; he had never liked anything less. What could he do, what could he say? If the girl were irreclaimable could he pretend to like it? To attempt to reclaim her was permissible only if the attempt should succeed. To try to persuade her of anything sordid or sinister in the man to whose deep art she had succumbed would be decently discreet only in the event of her being persuaded. Otherwise he should simply have damned himself. It cost him an equal effort to speak his thought and to dissemble; he could neither assent with sincerity nor protest with hope. Meanwhile he knew--or rather he supposed--that the affianced pair were daily renewing their mutual vows. Osmond at this moment showed himself little at Palazzo Crescentini; but Isabel met him every day elsewhere, as she was free to do after their engagement had been made public. She had taken a carriage by the month, so as not to be indebted to her aunt for the means of pursuing a course of which Mrs. Touchett disapproved, and she drove in the morning to the Cascine. This suburban wilderness, during the early hours, was void of all intruders, and our young lady, joined by her lover in its quietest part, strolled with him a while through the grey Italian shade and listened to the nightingales. | Summary: Isabel goes to breakfast and prepares to tell Mrs. Touchett the shocking news. Mrs. Touchett, not one to be shocked, already knows - apparently, with her innate meddling radar, she could just feel it in the air. She disapproves, and blames Madame Merle for betraying her. Isabel doesn't see what Madame Merle has to do with anything. Mrs. Touchett complains that there is no reason to marry Osmond; he has neither name nor fortune. Isabel rather snootily responds that she doesn't need to explain herself, and, even if she wanted to, she wouldn't know how. Yeah, that sounds like love to us. Mrs. Touchett asks Isabel whether Ralph would have changed her mind. Isabel says that Ralph would disagree with anyone she chose to marry. Isabel tells Mrs. Touchett about Caspar visiting and leaving within one day. Two days later, Ralph arrives. Isabel waits for Mrs. Touchett to tell him, and for Ralph to bring up the engagement with her, but he does not. Ralph is hurt and saddened - the person he found the most intriguing has let him down. Ralph hopes to dissuade Isabel from going through with her engagement. Osmond has been meeting up with Isabel outside of Palazzo Crescentini every day. They walk in a quiet park together. | 3,484 | 295 | booksum | en |
Summarize: [0001] This application claims priority to U.S. Provisional Patent Application Ser. No. 61/539,406 filed on Sep. 26, 2011, and incorporated herein in its entirety by reference. [0002] The disclosed device relates to infusion ports. More particularly, it relates to an interface covering for infusion ports which are conventionally implanted subcutaneously in a patient's body. The device and method herein provides an interface between the port exterior surfaces and the patient's surrounding tissue. The film interface helps prevent infections, patient discomfort, and significantly eases the task of removing the port by inhibiting tissue growth upon the port surfaces. FIELD OF THE INVENTION Background of the Invention [0003] Subcutaneous infusion ports are a preferred form of long term central venous access for patients treated by oncology departments, and for patients requiring long term direct injection of medications. The implanting and use of such ports are preferred, because they provide ease of access for the medical professional for infusion of drugs and are concurrently a less painful and less intrusive manner of infusing those drugs to the patient. [0004] Further, because the port employed to receive and communicate drugs and fluids to the patient is subcutaneous, there is a generally significantly lower infection rate long term versus the employment of multiple or continual incisions to yield required injection sites which are necessary when not using such ports. [0005] Infusion ports have several other advantages over other methods of venous access. One advantage is the ability to implant such ports under local anesthesia as an outpatient procedure, thereby reducing costs to the patient. Another is the long term comfort of the patient who need not endure numerous surgeries to provide required venous access on numerous occasions. [0006] During the implantation process, a surgeon, by forming a skin incision, creates a superficial infusion port pocket under the skin of the patient and above the surrounding flesh or muscle. The port pocket provides a secure position to place the port in a location determined to provide for access to the appropriate blood vessel using a tube or catheter which exits the port. Once the port is implanted, the incision is sutured. [0007] However, such ports can be left implanted for days, weeks, or longer, and such long term implantation of such ports is not without complications. First, the creation of large profile chambers or pockets between the patient's skin and surrounding flesh, over time can cause skin erosions. Such erosions can be complicated by any infection from pathogens which may have accidentally left on the port exterior surface, or from a reaction of the patient's immune system in the tissue surrounding the port caused by its presence. [0008] Additionally, cell growth occurs over time within the pocket for the injection port. Since cells seek attachment to other cells or a support surface this generally causes cells to become attached to the exterior of the implanted port over time. Such a cell ingrowth makes it significantly more difficult to remove the infusion port from the patient. This is because cells in the tissue of the surrounding structures forming the pocket will attach to the exterior of the implanted port due to such cell growth thereon and therebetween. [0009] For the patient this cell growth and attachment generally becomes uncomfortable, especially if accompanied by infection. For the surgeon, it causes the need for a debridement of the cells from the port upon removal of the port from its implant site. Because the debridement must be carefully done to avoid blood vessels, it increases the time for the procedure and the ultimate pain the patient suffers after removal. [0010] Such cell growth is additionally a problem in that the apertures, which are provided on the body of such ports, for the surgeon to suture the port in position under the skin of the patient, must be filled with a material in order to prevent cell growth into the apertures of the port body. Currently, silicone plugs are positioned in the suturing apertures in a timely manufacturing or operative procedure. [0011] Because of the filling of the apertures with a plastic or silicone material which cures to a hardened material, surgeons must, during implantation of a port, use significantly more hand and finger pressure and strength to generate it. This is required to force the needle and suture through the cured material positioned within the apertures in the port while suturing the port into position in an implantation pocket. [0012] As such, there exists a continual unmet need for a means for a medical professional to retard or prevent cell ingrowth and overgrowth upon the exterior surfaces of implanted infusion ports and the like. Such a device should be easily engaged to the exterior surface of the implant in a sterile environment. Such a device should prevent, or significantly impair the ingrowth or overgrowth of cells upon the port exterior surface during the duration of implantation in the patient to aid in an easy removal. Finally, such a device should eliminate the need to fill suturing holes or apertures in the port body, with cured plastic material, which subsequently increases the surgeon's effort and time during implantation and suturing therethrough. [0013] With respect to the above, before explaining at least one preferred embodiment of the invention in detail or in general, it is to be understood that the invention is not limited in its application to the details of construction and to the arrangement of the components or the steps set forth in the following description or illustrated in the drawings. The various apparatus and methods of the invention are capable of other embodiments, and of being practiced and carried out in various ways, all of which will be obvious to those skilled in the art once the information herein is reviewed. 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. [0014] As a consequence, those skilled in the art will appreciate that the conception upon which this disclosure is based may readily be utilized as a basis for designing and producing interface covers to fit on infusion and other implantable ports used for long term delivery of drugs and medicine to patients and the like, and for carrying out the several purposes of the present disclosed device and method. It is important, therefore, that the embodiments, objects and claims herein, be regarded as including such equivalent construction and methodology insofar as they do not depart from the spirit and scope of the present invention. SUMMARY OF THE INVENTION [0015] The interface cover device herein is configured for employment engaged over and in combination with an injection port (or portacath) which is a small medical appliance that is conventionally installed in a formed pocket beneath the skin of a patient. Such ports are designed for implantation under the skin of a patient using an incision which is then sutured. [0016] Such infusion ports conventionally have a septum or membrane cover above an interior reservoir which is positioned on an upper surface of the port which in this case is closest to the patient's skin. This septum provides a self-sealing means to communicate with an underlying reservoir. It is adapted to be pierced multiple times by a needle or other means to communicate fluid, medicine or drugs into the underlying reservoir or for the taking of blood samples therefrom on numerous occasions. [0017] A catheter, or other means for sealed communication of a lumen between a blood vessel, and the interior reservoir connects the reservoir under the septum to an internal blood vessel such as a vein or artery. [0018] It is the exterior surface of the body of the port, and also the septum, which will suffer from cell ingrowth during the duration of the implantation. Further, it is this same surface that can carry pathogens into the body of the patient if improperly sterilized or contaminated somehow prior to implant. [0019] The interface cover device herein, allowing the method herein, is formed of a thin film. The film sheet is configured to engage over such an implantable port and to cover a majority of its external surface area in an as-used position or engagement. Thus, in the as-used position, with the film sheet engaged upon and covering such a port, the device provides a separation or barrier between surrounding tissue and the surface of the port and a means to prevent cell ingrowth and overgrowth on the implanted port and its surfaces. [0020] The film sheet may be custom cut and dimensioned to fold and attach to the exterior surface of the port. In the simplest form of the device, the film sheet is folded around the entire port and adhered using adhesive or a shrinking by heat and resulting compression, or similar means for adhesion. In other preferred modes, the film sheet is enhanced using multiple materials such as a heat-shrinking polymeric material which contacts the port surface on a first side and has a cell growth inhibiting material such as a silicone or titanium layer or coating or surface on the opposite or second side. [0021] The entire film sheet, or the exterior facing surface, is formed as noted of a material that inhibits cell growth and/or cell attachment to the underlying port and its surfaces. The inhibiting material can be one or a combination of materials from a group including silicone, nano silicone, titanium, PTFE, silver ions, or other flexible surface materials which will cause cell growth limiting substances in addition to the action of the silicone film forming the device, and/or infused with medicines to prevent infection such as antibiotics which would be delivered slowly and directly into the pocket formed for the implant where infection is a frequent problem. [0022] Engaged over the body of such a port, the device provides a cover over the open apertures employed for suturing the port in place in the pocket formed in the tissue under a patient's skin. This cover allows for the suture apertures to be left open rather than filled. Thereby making it easier for the surgeon to suture through the open ports to mount the port in place. [0023] The device would be formed in a sheet which is configured to form fit the contours of the implanted port and may be formed to cover all but the septum in one mode, or as a donut shape in another which would leave the septum and/or lower surface exposed. Additionally, the film sheet may be formed of heat-shrink material at the first surface with a silicone layer or surfacing on the opposite surface, using a material such as Olefin polymers with a layer of silicone such as the film CLYSAR HPS which is manufactured by the Dupont company. Employing a film sheet with the silicone exterior or second surface, the port may be wrapped in the film and exposed to heat which will shrink it to the conforms of the port which the sheet maintains once the heat is removed. This is a particularly preferred mode of the device and method herein since it provides the most contoured fit of the film to the port thereby forming a silicone barrier between the port surface and the tissue of the formed pocket it occupies. [0024] If provided with heat-shrink film with a silicone or other exterior surface, the sheets will shrink to fit so long as they are reasonably formed to the correct size. With the addition of an adhesive to the first surface contacting the port, adhesion is enhanced also. The film sheets may be provided in sterile envelopes or from containers of dispensable sheets, which are maintained sterile until employed and are configured to engage over the port and shrink to fit and adhere to it. [0025] The foregoing has outlined rather broadly the more pertinent and important features of the device and method herein employing a film formed to cover and interface between an infusion port or other implanted device and the patient's cells in the pocket surrounding it on an implant in order that the detailed description of the invention that follows may be better understood so that the present contribution to the art may be more fully appreciated. Additional features of the invention may be described hereinafter which form the subject of the claims of the invention. It should be appreciated by those skilled in the art that the conception and the disclosed specific embodiments may be readily utilized as a basis for modifying or designing other interface coverings for implanted ports for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions and methods do not depart from the spirit and scope of the invention as set forth herein and are considered within the scope of this invention. [0026] Further, 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 arrangement 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. THE OBJECTS OF THE INVENTION [0027] It is therefore an object of the present invention to provide a cover and interface between the exterior surfaces of an implanted infusion port or other implanted device and surrounding tissue during the term of implantation. [0028] It is another object of this invention to provide such a device and method that may be easily incorporated for engagement over existing implantable ports and be easily interfaced with the installed base of medical equipment at medical facilities through the use of film sheets which are configured to conform, and/or shrink to conform to the exterior contours and surfaces of the implanted port. [0029] The foregoing has outlined some of the more pertinent objects of the invention. These objects should be construed to be merely illustrative of some of the more prominent features and applications of the intended invention. Many other beneficial results can be attained by applying the disclosed method and port interface device in a different manner or by modifying the invention within the scope of the disclosure. Accordingly, other objects and a fuller understanding of the invention may be had by referring to the summary of the invention and the detailed description of the preferred embodiment in addition to the scope of the invention defined by the claims taken in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS [0030] The accompanying drawings, which are incorporated herein and form a part of this specification, illustrate embodiments of the invention and together with the detailed description, serve to explain the principles of this invention. [0031] FIG. 1 depicts a perspective view of a conventional infusion port device employable with the film interface herein. [0032] FIG. 2 depicts a top view of the device of FIG. 1, showing the septum surface of the implantable port and surrounding top surface. [0033] FIG. 3 depicts a side view thereof. [0034] FIG. 4 depicts a bottom view of a port such as in FIG. 1. [0035] FIG. 5 depicts a side view of the device herein showing the film sheet of material defining the cover formed to engage over the conventional port of FIGS. 1-4. [0036] FIG. 6 depicts a view of the device herein showing the film sheet engaged on a port of FIG. 3. [0037] FIG. 7 depicts a top view of a conventional port as in FIG. 2, with the device engaged to cover the port surfaces surrounding the septum. [0038] FIG. 8 depicts a mode the device wherein the film sheet is configured for a wrapping and covering of the entire surface of the port. [0039] FIG. 9 depicts a second mode of the bottom of the interface device herein, having an elastic center aperture employable for engaging the flexible film material over the port it surrounds. [0040] FIG. 10 depicts a container for holding and dispensing the properly configured film sheets during implantation. [0041] FIG. 11 depicts an envelope or package employed to maintain the film sheet material within, sterile until engaged to the port. [0042] FIG. 12 depicts the film sheet having an optional aperture for surrounding the septum and having a first side surface for contact and adherence to the underlying port, and a second surface layered or coated or infused with a cell growth retardant and/or pathogen growth inhibitant. [0043] FIG. 13 depicts a the film sheet from FIG. 12 formed to a three-dimensional oversized covering in a shape configured to engage over the contours of the body of the port after insertion and using a heat source to shrink the film, with optionally adhesive on the first surface. [0044] FIG. 14 shows the film sheet adhered and contoured to the bottom surface of the infusion port. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS [0045] Referring now to the drawings 1 - 14, wherein similar parts of the invention are identified by like reference numerals, there is shown various views of a conventional prior art pressure port 11 in FIGS. 1-4. Such ports are conventionally implanted, as noted above, under the skin of a patient in pockets formed between overlying skin and surrounding tissues. [0046] The device 10 and method herein employs a flexible film 13 sheet as shown in FIG. 12 which has a first side surface 15 and an opposite second side surface 17 formed as a single layer or laminate. The dimensions of the film 13 sheet are configured to fully cover the desired exterior surfaces of a conventional port 11 as in FIGS. 1-5. If the material forming the film 13 is heat-shrinkable material, which will shrink and conform to the port exterior surfaces when heated, then the dimensions of the sheet of film 13 need to accommodate the amount of shrinkage and fully cover all or the intended exterior surface of the port 11. A means for imparting heat to shrink the film 13 sheet once engaged to the port 11 could be a blow-dryer however a UV Light source would be more preferred due to the fact it does not disrupt air in the sterile field of the procedure, and, UV light will kill most pathogens should they have somehow gotten on the surface of the port 11 or the film 13. [0047] As shown in FIG. 5 which shows the device 10 formed by the method herein, from a side view, the formed device 10 employs the film 13 sheet. The entire sheet of film 13 may be formed as a single film layer of material which inhibits cell growth and/or cell attachment to the underlying port 11 and its surfaces, or the second surface 17 may be have such cell growth inhibiting material splattered, sprayed, coated, laminated or otherwise placed upon and covering substantially the entire second surface 17. Such cell growth inhibiting material can include one or a combination of materials, from a group of cell growth inhibiting materials, including silicone, nano silicone, titanium, PTFE, and silver ions. Currently one or a combination of silicone or nano silicone is particularly preferred for forming, or covering the second surface 17 as experimentation has shown silicone is inert as to causing cell irritation and is an exceptional inhibitant of cell growth. Titanium is also an excellent material for the same reasons and because it enhances the lubricity of the area surrounding the implant. [0048] The film 13, either planar or formed as a three-dimensional covering, has the second surface 17 which will contact surrounding tissue when the port 11 is implanted, and provide a barrier between the cells and tissue and any adhesion to the exterior surface of the port 11. Of course the second surface 17 of the film can be made of, coated with, laminated with, deposited with, or impregnated with, other materials which inhibit cell growth would occur to those skilled in the art, and such is anticipated within the scope of this invention. [0049] In FIG. 6 is shown the assembled device 10 with the film 13 such as shown in FIG. 12 or 13, is engaged and conformed to the installed as-used position covering the port 11. If the optional aperture 16 is provided the film 13 can be engaged to leave the septum 12 uncovered to provide uninhibited access for injections. If the aperture 16 is to be employed, it would be formed in either the planar sheet 13 of FIG. 12 or the contoured sheet 13 of FIG. 13 in a size to cover the port 11 surface surrounding the septum 12 when the film 13 is operatively engaged in the as-used position. [0050] To that end, the device 10 can either use film 13 which is either molded or cut or thermoformed, or otherwise formed to such configuration, with an aperture 16 sized to engage around the septum 12 and to allow needle access to the septum 12. If a heat shrink material is not employed, in a similar fashion an elastic surrounded or formed aperture 16 can be formed to stretch over the perimeter of the port 11, and allow for installation of the film 13 sheet on the port 11 much like a sock is engaged to a foot. This elastic mode of the film 13 might be combined with a means for imparting heat to the film such as a UV lamp, and provide for a shrinking of the film 13 to a tight fit and adhesive or other contact of the first surface 15 with the exterior surface of the port 11 surrounding the septum 12. [0051] FIG. 7 shows the assembled device 10 with the film 13 installed to cover the body of the port 11, and with the aperture 16 in a registered engagement to allow access to the septum 12. Also shown is the film 13 covering the suture apertures 18 protecting them from cell ingrowth, while allowing the elimination of the hard plugs therein which conventionally inhibit the surgeon's suturing. [0052] FIG. 8 depicts the assembled device 10 with the film 13 having its first surface 15 engaged and covering the entire bottom exterior surface of the port 11. Mounting of the film 13 to the port 11 is accomplished, as noted, using a means for heating and heat-shrink film 13 which when wrapped around the port 11 from the planar sheet of FIG. 12, or engaged using the dimensioned sheet of FIG. 13, will shrink and adhere to the exterior of the port 11. Adherence to the exterior surface of the port 11 may be with one or a combination of an adhesive or attachment of the film 13 itself. [0053] Alternatively, if the film 13 is configured in the dimensioned shape such as that of FIG. 13, which is slightly larger than the port 11 but in the same general shape, the threaded end 33 of the port can be inserted through the septum aperture 16, and exited through a side aperture 19 and the configured film 13, forming the device 10, will contact the exterior surfaces of the port 11 when heated to shrink, and adhere thereto using adhesive positioned on the first surface 15 or just by contact with the first surface 15. Or if the film 13 is elastic in nature, a sliding of the port 11 and threading through the two apertures in the above referenced manner will allow the film 13 to then retract and engage upon the exterior of the port 11. [0054] FIG. 9 depicts a second mode of the bottom of the assembled device 10 herein, having a center aperture 16 employable for engaging the port 11 with the film 13 of the assembled device 10. This formation would place a small opening where the aperture 16 is positioned, in another mode of installation. [0055] FIG. 10 depicts a container 31 for holding and dispensing the properly configured film 13 during implantation. If in a planar sheet 13 such as in FIG. 12, it may be dispensed directly, or using a removable cover 35. Or as shown in FIG. 11 depicts an envelope or removable cover 35 can be employed and packed with the port 11 kit, to maintain the sterility of the film 13 until engaged on the port 11. The film 13 can be positioned in the cover 35 or container 31 in either the cut planar sheet mode of the film 13 of FIG. 12, or the contoured sheet mode of the film 13 of FIG. 13. [0056] Once removed from the container 31 and or cover 35, the film 13 is engaged upon the port 11 in a manner consistent with the mode of provision of the sheet 13. If planar as in FIG. 12 the film 13 is wrapped around the port 11 as in FIG. 12 and adhered thereto using adhesive or the attraction of the first side surface 15 to the port surface. If the film is provided in a dimensioned form as in FIG. 13, using either elastic film 13 which will contract and engage the port 11, or as a heat shrinking film which is exposed to a UV source or other means to heat the film 13, it too can be provided in a container 31 and/or the cover 35 to maintain sterility till engaged to the port 11. [0057] FIG. 12 as noted depicts the film 13 in a sheet having an optional aperture for surrounding the septum 12 and having a first side surface 15 for contact and adherence to the underlying port 11. Also shown is the second surface 17 layered or coated or infused with a cell growth retardant and/or pathogen growth inhibitant noted herein. [0058] FIG. 13 depicts the film 13 sheet from FIG. 12 formed in the three dimensional configuration, which substantially follows to the shape of the exterior surface of the port 11. As noted it may be elastic film 13, or as depicted, formed oversized and then reduced in size using a heat source 39 such as a UV light or hot air, to cover the exterior contours of the exterior surface of the port 11. Engagement with the surface can be as noted one or a combination of adhesion means such as compression in a heat shrink process and/or adhesive, and or an ionic attraction of the first surface 15 to the surface of the port 11. [0059] FIG. 14 shows the film 13 sheet from FIG. 12 or 13, adhered against and the bottom surface of the infusion port 11 and continuing up the contours of the sides of the port 11. [0060] As such, the system and device 10 provided by the engagement of the film 13 sheet to cover the all or substantially all of the exterior surface of an implanted port 11, provides great utility to the surgeon and patient by decreasing cell ingrowth which is hard to remove and decreasing irritation and infection while implanted. Using the film 13 which is sized to envelop all or most of the exterior surface of the port 11 and adhere thereto on a first side surface of the film, the film 13 in this as-used position provides a barrier to cell ingrowth to the exterior of the port 1. [0061] Means for engagement may be my forming the sheet 13 similar in configuration but larger than the exterior of the port 11 and using a heat source to shrink the film, or by using an elastic film sized equal to or slightly larger than the area of the exterior of the port 11, and stretching the film 11 thereover. Or, the film 13 can be provided as a pre-cut planar sheet which may be wrapped to envelop most of the exterior surface of the port 11. Or, as noted the film 13 may be formed in a three dimensional configuration larger than the exterior of the port 11 and then shrunk to a compressive and/or adhesive engagement of the first surface 15 with the exterior of the port 11. [0062] In this as-used configuration with the first surface 15 of the film 13 in a contact and engaging the exterior of the port 11, the primary object of presenting a second surface 17 of the film 13 provides for contact with surrounding cells as a barrier to cell ingrowth is accomplished. Further in all modes the second surface 17 can be a laminate or spray coated, or sputter coated, surface of one or a combination of cell ingrowth inhibitors such as silicone and titanium or any of the aforementioned cell growth inhibitors. [0063] While all of the fundamental characteristics and features of the disclosed port interface device and method herein have been described herein, with reference to particular embodiments thereof, a latitude of modification, various changes and substitutions are intended in the foregoing disclosure and it will be apparent that in some instance, some features of the invention will be employed without a corresponding use of other features without departing from the scope of the invention as set forth. It should be understood that such substitutions, modifications, and variations may be made by those skilled in the art without departing from the spirit or scope of the invention. Consequently, all such modifications and variations are included within the scope of the invention as defined herein. | Summary: An apparatus and method for positioning a barrier to cell ingrowth on the exterior surface of a port being implanted under the skin of a patient is provided. The barrier is formed by a film sheet configured to form a cover of the exterior surface of the port and remain mounted thereon in an as-used position. So engaged the film forms a barrier to cell ingrowth upon the port and additionally positions cell growth inhibitors on the exposed surface of the film adjacent to the surrounding cells. Engagement of the film to the port is accomplished by one or a combination of elastic contraction, heat shrinking or adhesive. | 6,794 | 126 | big_patent | en |
Summarize: By. Associated Press Reporter. A mother who starved her four young. sons and kept them in a filthy Denver apartment covered in feces could. spend up to seven years in prison after pleading guilty Friday to her. second offense of child abuse. Prosecutors. said the case involving Lorinda Bailey, 36, was among the most horrific. they had ever seen, but the state's child abuse laws kept them from. pursuing harsher penalties because the children, ages 2 to 6, did not. suffer serious physical injuries. Six other charges were dropped in exchange for Bailey's guilty plea to a single felony count. Nightmare mom: Lorinda Bailey, 36, seen here in 2013, pleaded guilty to a second offense of child abuse on Friday in exchange, six other counts are being dropped. 'The way the child abuse statue reads,. this was a disposition we thought would hold her accountable as best we. could under the law,' said Lynn Kimbrough, a spokeswoman for the Denver. District Attorney's Office. Police say Bailey and the boy's father, Wayne Sperling, 66, kept their sons in an home filled with cat excrement and buzzing with flies. The. children were not potty-trained and could communicate only in grunts. when authorities removed them from the home in October. They were. alerted by an emergency room doctor, who noticed that the youngest boy. was unwashed, reeked of cigarette smoke and had bruises consistent with. pinching. Doctors couldn’t find any medical records at all for the five-year-old. Wayne Sperling, left, and Lorinda Bailey, right, are accused of neglecting their four young boys and keeping them in a filthy Denver apartment full of cat feces and flies. 'Sick': In 2006, Bailey's children were found covered in dirt and hungry after neighbors saw them playing in the road. She and Sperling were put on probation and ordered to take parenting classes. Monstrous: Officials said that Bailey and Sperling's treatment of their children was the worst case of abuse they have every seen. Sperling. told investigators he was the primary guardian for the boys. Bailey. said she was living in another apartment in the same building but. saw the boys most days. The elderly father has. pleaded not guilty and faces a hearing in October. Bailey remains. free on bond pending her sentencing. Neighbors. said they previously called social services with concerns about the. family, but the agency would not publicly discuss the case for. confidentiality reasons. The couple lost custody of other children amid similar allegations. Officers. found rotten food, trash and insects in the apartment in October 2006,. after passers-by reported two young children playing in the street. The. children mostly grunted and pointed to communicate. Bailey and Sperling pleaded guilty in June 2007 to misdemeanor child abuse and lost custody of their three oldest children. The latest case warranted felony charges because it was a repeat child abuse offense, Kimbrough said. The four boys are now improving in foster care and living together, she said. Charged: Sperling and Bailey were arrested last October and charged with child abuse after their sons were found in filthy conditions. Scene: A man walks past the residence of Sperling, where the four boys were found in deplorable conditions. Police records show that officers had previously responded to complaints | Summary: Lorinda Bailey, 36, pleaded guilty to a single count of abuse in exchange for having six other charges dropped. Bailey's husband Wayne Sperling, 66, pleaded not guilty in the case. Their four sons were found in deplorable conditions in Sperling's home which was full of cat feces and flies. The boys, aged two to six, could communicate only in grunts, were malnourished and weren't toilet trained. | 823 | 109 | cnn_dailymail | en |
Write a title and summarize: Despite the recent progress in sequencing technologies, genome-wide association studies (GWAS) remain limited by a statistical-power issue: many polymorphisms contribute little to common trait variation and therefore escape detection. The small contribution sometimes corresponds to incomplete penetrance, which may result from probabilistic effects on molecular regulations. In such cases, genetic mapping may benefit from the wealth of data produced by single-cell technologies. We present here the development of a novel genetic mapping method that allows to scan genomes for single-cell Probabilistic Trait Loci that modify the statistical properties of cellular-level quantitative traits. Phenotypic values are acquired on thousands of individual cells, and genetic association is obtained from a multivariate analysis of a matrix of Kantorovich distances. No prior assumption is required on the mode of action of the genetic loci involved and, by exploiting all single-cell values, the method can reveal non-deterministic effects. Using both simulations and yeast experimental datasets, we show that it can detect linkages that are missed by classical genetic mapping. A probabilistic effect of a single SNP on cell shape was detected and validated. The method also detected a novel locus associated with elevated gene expression noise of the yeast galactose regulon. Our results illustrate how single-cell technologies can be exploited to improve the genetic dissection of certain common traits. The method is available as an open source R package called ptlmapper. Modern genetics aims to identify DNA variants contributing to common trait variation between individuals. A high motivation to map such variants is shared worldwide because many heritable traits relate to social and economical preoccupations, such as human health or agronomical and industrial yields. In addition to the molecular knowledge they provide, these variants fuel the development of personalized and predictive medicine as well as the improvement of economically-relevant plants, animal breeds or biotechnology materials. However, this high ambition is accompanied by a major challenge: common traits are under the control of numerous variants that each contribute little to phenotypic variation [1], and this modest contribution of each variant hampers the statistical power to detect them. Power is further limited by the multiplicity of linkage tests when scanning whole genomes. The consequence of this has been debated under the term" missing heritability" : most of the genetic variants of interest remain to be identified. Currently, this issue is handled by modelling the effect of known or hidden factors, and by scaling up sample size up to tens of thousands of individuals [2–4]. Practically, however, cohort size cannot be infinitely increased, and relevant factors are difficult to choose. Studies would therefore greatly benefit from a better detection of small genetic effects, and from a reduction of the number of genomic loci to test. Small-effect variants are typically associated with predisposition (or incomplete penetrance): carriers of a mutation display a phenotype at increased frequency, but not all of them do. In this probabilistic context, the statistical properties of cellular traits may sometimes become informative: a tissue may break because cells have an increased probability to detach, a tumor may emerge because a cell type has an increased probability of somatic mutations, a chemotherapy may fail if cancer cells have an increased probability to be in a persistent state. In other words, molecular events in one or few cells can have devastating consequences at the multicellular level. As discussed previously [5], cellular-scale probabilities are likely related to the genotype and this relation may sometimes underlie genetic predisposition [6]. Striking examples are genetic factors affecting the mutation rate of somatic divisions and thereby modifying cancer predisposition. These loci have a probabilistic effect on a cellular trait: the amount of de novo mutations in the cell' s daughter. Other loci may modulate the heterogeneity between isogenic cancer cells that underlies tumour progression [7,8] and resistance to chemotherapy [9–11]. They would then change the fraction of problematic cells between individuals and thereby disease progression or treatment outcome. Fortunately, the experimental throughput of single-cell measurements has recently exploded. Technological developments in high-throughput flow cytometry [12], multiplexed mass-cytometry [13], image content analysis [14–16] and droplet-based single-cell transcriptome profiling [17,18] now offer the possibility to estimate empirically the statistical distribution of numerous molecular and cellular single-cell quantitative traits. We therefore propose to scan genomes for variants that modify single-cell traits in a probabilistic manner, which we call single-cell Probabilistic Trait Loci (scPTL). This requires to monitor not only the macroscopic trait of many individuals but also a relevant cellular trait in many cells of these individuals. After scPTL are found, they can constitute a set of candidate loci to be directly tested for a possible small effect on the macroscopic trait of interest. Methods are needed to detect scPTL. With its fast generation time, high recombination rate and reduced genome size, the unicellular yeast Saccharomyces cerevisiae offers a powerful experimental framework for developing such methods. Using this model organism, scPTL were discovered by treating one statistical property of the single-cell trait, such as its variance in the population of cells, as a quantitative trait and by applying Quantitative Trait Locus (QTL) mapping to it [19,20]. However, this approach is limited because it is difficult to anticipate a priori which summary statistics must be used. We present here the development of a genome-scan method that exploits all single-cell values with no prior simplification of the cell population phenotype. Using simulations and existing single-cell data from yeast, we show that it can detect genetic effects that were missed by conventional linkage analysis. When applied to a novel experimental dataset, the method detected a locus of the yeast genome where natural polymorphism modifies cell-to-cell variability of the activation of the GAL regulon. This work shows how single-cell quantitative data can be exploited to detect probabilistic effects of DNA variants. Our approach is conceptually and methodologically novel in quantitative genetics. Although we validated it using a unicellular organism, it opens alternative ways to apprehend the genetic predisposition of multicellular organisms to certain complex traits. We specify here the concepts and definitions that are used in the present study. Let X be a quantitative trait that can be measured at the level of individual cells. X is affected by the genotype of the cells and by their environmental context. However, even for isogenic cells sharing a common, supposedly homogeneous environment, X may differ between the cells. To describe the values of X among cells sharing a common genotype and environment, we define a single-cell quantitative trait density function f [5] as the function underlying the probability that a cell expresses X at a given level (Fig 1A). Statistically speaking, f represents the probability density function of the random variable X. In the present study, this function f (X) constitutes the' phenotype' of the individual from whom the cells are studied. As for any macroscopic phenotype, it can depend on the environmental context of the individual (diet, age, disease…) as well as on its genotype. Single-cell trait density functions also obviously depend on the properties of the cells that are studied, such as their differentiation state or proliferation rate. We focus here on the effect of the genotype. Conceptually, cells from one individual may follow a density function of X that is different from the one followed by cells of another individual, because of genotypic differences between the two individuals (Fig 1B). The important concept is that the genetic difference has probabilistic consequences: it changes the probability that a cell expresses X at a given level, but it does not necessarily change X in most of the cells. Depending on the nature of trait X and how the two functions differ, such a genetic effect can have implications on macroscopic traits and predisposition to disease [5]. The term single-cell Probabilistic Trait Locus will refer here to a genetic locus modifying any characteristics of f (that is, changing allele A in allele B at the locus changes the density function f of X, i. e. fB ≠ fA). A quantitative trait locus (QTL) linked to X is a location on a chromosome where a genetic variant changes the mean or the median of X in the cell population. Similarly, a varQTL is a genetic locus changing the variance of X and a cvQTL is a genetic locus changing the coefficient of variation (standard deviation divided by the mean, abbreviated CV) of X in the cell population. All three types of loci (QTL, varQTL and cvQTL) assume a change in f and they are therefore special cases of scPTL. However, not all scPTL are QTL: many properties of f may change while preserving its mean, median, variance or CV. The purpose of the present study was to develop an approach that could identify scPTL without knowing a priori how it might change f. An important question before investing efforts in scPTL mapping is whether genotypes can modify f without affecting its expected value (the mean of X). If not, then QTL mapping will capture the genetic modifiers of f and searching for more complex scPTL is not justified. In contrast, if other-than-mean genotypic changes of f are frequent, then scPTL can considerably complement QTL to control single-cell traits. In this case, scPTL mapping becomes important. In multicellular organisms, cell types and intermediate differentiation states constitute the predominant source of cellular trait variation. Studying their single-cell statistical characteristics requires accounting for the developmental status of the cells. This constitutes a major challenge that can be avoided by studying unicellular organisms. The yeast S. cerevisiae provides the opportunity to study individual cells that all belong to a single cell type, in the context of a powerful genetic experimental system. By analysing specific gene expression traits in this organism, we and others identified loci that meet the definition of scPTL but not of QTL [20,21]. This illustrated that, for some traits, scPTL mapping could complement classic quantitative genetics to identify the genetic sources of cellular trait variation. To estimate if non-QTL scPTL are frequent, we re-analysed an experimental dataset corresponding to the genetic segregation of many single-cell traits in a yeast cross (Fig 2A). After a round of meiosis involving two unrelated natural backgrounds of S. cerevisiae, individual segregants had been amplified by mitotic (clonal) divisions and traits of cellular morphology were acquired by semi-automated fluorescent microscopy and image analysis [22]. This way, for each of 59 segregants, 220 single-cell traits were measured in about 200 isogenic cells, which enabled QTL mapping of these traits. We reasoned that if all scPTL of a trait are also QTL, then a high genetic heritability of any property of f should coincide with a high genetic heritability of the expected value of f. In particular, the coefficient of variation (CV) of a single-cell trait should then display high heritability only if the mean value of the trait also does. To see if this was the case, we computed for each trait the broad-sense genetic heritabilities of both the mean and CV of the trait. Note that the genetic heritability computed here is not the same as the mitotic heritability of cellular traits transmitted from mother to daughter cells. Here, a value (mean or CV) is computed on a population of cells, and its heritability corresponds to the proportion of its variation that can be attributed to genetic differences between the cell populations (see methods). Overall, heritability of mean was higher than heritability of CV, and the two types of heritabilities were correlated (Fig 2B). We also observed that several traits had high heritability of CV and low heritability of their mean value, or vice versa. This indicates that, for some traits, genetic factors exist that modify the trait CV but not the trait mean. This observation is in agreement with the complex CV-vs-mean dependency previously reported in this type of data [23,24]. We therefore sought to develop a method that can detect scPTL that do not necessarily correspond to QTL. One way to identify scPTL from experimental measures is to compute a summary statistic of the trait distribution, such as one of its moments, and then scan for QTL controlling this quantity. This approach is particularly appropriate when searching for specific genetic effects on f, such as a change in the level of cell-to-cell variability, and a few previous studies successfully used it to map varQTL and cvQTL [19,20,22,25,26]. However, it is less adapted when nothing is known on the way f may depend on genetic factors. Scanning for scPTL considers the entire distribution of single-cell trait values as the phenotype of interest and searches the genome for a statistical association with any change in the distribution. We assume that for a set of genotypic categories (individuals for multicellular organisms, or populations of cells for unicellular ones), a cellular trait has been quantified in many individual cells of the same type. This way, the observed distribution of the trait constitutes the phenotypic measure of individuals. We also assume that a genetic map is available and the individuals have been genotyped at marker positions on the map. The method we propose is based on three steps. First, a distance is computed for all pairs of individuals in order to quantify how much their phenotype differs. We chose the Kantorovich metric (also known as the Wasserstein distance or the earth-mover' s distance) to measure this distance because, unlike the Kullback-Leibler divergence, it satisfies the conditions of non-negativity, symmetry and triangle inequality and, unlike the Hellinger distance, it does not converge to a finite upper limit when the overlap between distributions diminishes [27]. The Kantorovich metric can be viewed as the minimum energy required to redistribute one heap of earth (one f-function) into another heap (a second f-function). It has enabled developments in various fields, ranging from mathematics [28] to economy (the minimal transportation problem) [29,30] to the detection of states from molecular dynamics data [27]. The next two steps are inspired from methods used in ecology, where spatial distinctions between groups are often searched after determining distances between individuals [31,32]. In step 2 of our method, individuals are placed in a vectorial space while preserving as best as possible the distance between them (Fig 3A). This is achieved by multi-dimensional scaling, a dimension-reduction algorithm [33]. The third step is the genetic linkage test itself. At every genetic marker available, a linear discriminant analysis is performed to interrogate if individuals of different genotypic classes occupy distinct sectors of the phenotypic space (Fig 3B and 3C). The optimal choice of dimensionality is determined dynamically and a permutation test assesses statistical significance in the context of the corresponding degrees of freedom. Note that if the dimensions have been reduced to a single one, then canonical analysis is not needed: the phenotypic value of each individual has become a scalar and linkage can be performed by standard QTL mapping. Finally, scPTL linkage is scored using the Wilks' lambda statistics. Statistical inference is made using empirical p-values produced by permutations where the identities of individuals are re-sampled. The full procedure is described in details in the methods section. We first evaluated if our method could detect scPTL from simulated datasets. To do this, we considered a probabilistic single-cell trait governed by a positive feedback of molecular regulations. This is representative of the expression level of a gene with positive autoregulation. As depicted in Fig 4A, the employed model is based on three parameters. For each individual, a set of parameter values was chosen and single-cell values of expression were generated by stochastic simulations. We chose to simulate a scPTL that modified the expected values of the parameters so that the skewness of cellular trait distribution is affected. To do so, we considered a panel of individuals and their genotype at 200 markers evenly spaced every 5cM. Parameter values of each individual were drawn from Gaussians and the mean of these Gaussians depended on the genotype at the central marker. This defined two sets of phenotypes that are depicted by blue and red histograms in Fig 4B. A universal noise term η was added to introduce intra-genotype inter-individual variation which, in real datasets, could originate from limited precision of measurements or from non-genetic biological differences between individuals. For each of five increasing values of η, about 130 individuals were simulated. We first scanned the generated dataset by QTL mapping, treating either the mean trait or its variance as the phenotype of interest. This way, the central scPTL locus was detected only when intra-genotype noise was null or very low (Fig 4C). This was anticipated because the mean and variance of the simulated trait values slightly differed between the two sets of individuals. In contrast, our new method allowed to robustly detect the scPTL locus even in the presence of high (up to 20%) intra-genotype noise (Fig 4D and 4E). The results described above using a simulated dataset suggest that the method can complement usual QTL mapping strategies. To explore if this was also the case when using real experimental data, we applied scPTL scans to the dataset of Nogami et al. [22] mentioned above (Fig 2A) where 220 single-cell traits were measured in about 200 cells from segregrants of a yeast cross. We applied three genome x phenome scans, each one at FDR = 10%. Two consisted of QTL interval mapping and were done by considering either the mean cellular trait value of the population of cells or the coefficient of variation of the cellular trait as the population-level quantitative trait to be mapped. The third scan was done using the novel method described here to map scPTL. Significant linkages obtained from this scan are available in S1 Table. As shown in Fig 5, the three methods produced complementary results. We detected more linkages with the scPTL method than with the 2 QTL scans combined (71 vs. 61 traits mapped). This illustrates the efficiency of using the full data (whole distribution) of the cell population rather than using a summary statistic (mean or CV). In addition, we expected that a fraction of scPTL would match QTL, because QTL controlling the mean or CV of cellular traits are specific types of scPTL. This was indeed the case, with 67% of scPTL corresponding to loci that were detected by at least one of the two QTL scans. For 11 cellular traits, a locus was found by QTL or cvQTL mapping but it was missed by the scPTL scan. This illustrates that the methods have different power and sensitivity. Importantly, 22 cellular traits were associated to scPTL that were not detected by the QTL search, suggesting that some probabilistic effects may affect poorly the trait' s mean or CV. Altogether, these observations highlight the complementarity of the different approaches and show that scPTL mapping can improve the detection of genetic variants governing the statistical properties of single-cell quantitative traits. Examples of scPTL of yeast cellular morphology are shown in Fig 6. One of the cellular traits measured was the distance between the center of the mother cell and the brightest point of DNA staining (Fig 6A). No QTL was found when searching genetic modifiers of the mean or CV of this trait, but a significant scPTL was mapped on chromosome II. When displaying trait distributions, it was apparent that segregants carrying the BY genotype at the locus had reduced cell-cell variability of the trait as compared to segregants having the RM genotype (Fig 6A, right panel). Consistently, a small cvQTL peak was seen on chromosome II, although this peak did not reach genome-wide statistical significance. This trait, which relates to the statistical properties of DNA migration during the early phase of cell division, provided a biological example where scPTL scan identified a genetic modulator of cell-to-cell variability that was missed by the QTL approach. Three other traits were of particular interest because they mapped to a position on chromosome VIII where a functional SNP was previously characterized in this cross. This SNP corresponds to a non-synonymous I->S mutation at position 469 in the Gα protein Gpa1p. It targets a domain that is essential for physical interaction with pheromone receptors Ste2p and Ste3p [34,35]. In the presence of pheromone, Gpa1p is released from the receptor and triggers a signalling cascade of molecular response that causes cell-cycle arrest and cell elongation (a process called' shmooing' ). In the absence of pheromone, improper binding of Gpa1pI469S to the receptor causes residual activation of the pathway in the BY strain, as seen by transcriptomic profiling [36], which explains why BY cells are more elongated [24] and proliferate slower [37] than RM cells. Here we saw that this locus is a scPTL, but not a QTL, of the degree to which cells are elliptical (Fig 6B). Displaying the distributions of this trait in each segregant revealed a remarkable amount of variability between the segregants, and that the BY allele at the locus corresponded to a modest reduction of the trait value as compared to the RM allele (sharper mode at slightly lower value). To see if this was due to the GPA1I469S mutation, we examined the data from a BY strain where this mutation was cured [22]. Remarkably, the single amino-acid substitution caused a mild but statistically significant redistribution of the trait values (Fig 6B). This change was comparable to the difference seen among the segregants, demonstrating the causality of the GPA1I469S SNP. Another trait, corresponding to the distance between the bud tip and the short axis of the mother cell, also mapped to this locus, with the RM allele associated to greater cell-cell variability, and data from the GPA1I469S allele-replaced strain validated this SNP as the causal polymorphism (Fig 6C). These observations suggest that either the residual activation of the pathway in absence of pheromone is not uniform among BY cells, or the proper inactivation of the pathway is not complete in all RM cells. This, and the fact that the mutation does not prevent BY cells from proliferating (as compared to pheromone-arrested cells), indicate that the detachment of Gpa1pI469S from the receptor is a rare event that has probabilistic effects on the cellular phenotype. Further investigations based on biochemistry, dynamic recording of individual cells and stochastic modelling are needed to understand how variation in binding affinity accounts for this effect. The results described here illustrate that scPTL scans can identify individual SNPs that modify single-cell trait distributions without necessarily affecting the trait mean. Finally, another trait corresponding to the angle of bud site position mapped to two scPTL loci and no QTL. One of these loci contained the GPA1 gene on chromosome VIII. Although the phenotype of bud site selection is not related to' shmooing', we examined if the GPA1I469S SNP was involved and found that it was not: the allele-replaced strain did not show a different trait distribution than its control (Fig 6D). Thus, other genetic polymorphisms at the locus should participate to the statistical properties of cellular morphology, by affecting the position of budding sites. We then explored if scPTL scanning could provide new results when applied to a molecular system that had been extensively characterized by classical genetics. The system we chose was the yeast GAL regulon which, in addition to be one of the best described regulatory network, presented several advantages. Natural strains of S. cerevisiae are known to display differences in its regulation [38,39] and the transcriptional response of cell populations can be tracked by flow cytometry. This provides data from large numbers of cells and therefore a good statistical power to compare single-cell trait distributions. In addition, acquisitions on many genotypes are possible using 96-well plates. We reasoned that if features of the cell population response segregate in the BY x RM cross (described above for morphology), then scPTL scanning might identify genetic variants having non-deterministic effects on the regulation of GAL genes. We first compared the dynamics of transcriptional activation of the network in the two strains BY and RM. This was done by integrating a PGal1-GFP reporter system in the genome of the strains, stimulating them by addition of galactose in the medium, and recording the response by flow cytometry. As shown in Fig 7, both strains responded and full activation of the cell population was reached after ~2 hours of induction. Interestingly, remarkable differences were observed between the two strains regarding the distribution of the cellular response. The BY strain showed a gradual increase of expression through time that was relatively homogeneous among the cells (unimodal distribution with relatively low variance), whereas the RM strain showed elevated cell-cell heterogeneity at intermediate activation time points (higher variance, with fraction of non-induced cells). This suggested that genetic polymorphisms between the strains might control the level of heterogeneity of the cellular response at these intermediate time points. We sought to map one or more of these genetic factors. To do so, we acquired the response of 60 meiotic segregants of the BY x RM cross. Using the data collected at each time point, we scanned the genome for scPTL of the reporter gene expression level using the novel genome-scan method described above. The procedure identified a locus on chromosome V position 350,744 that was highly significant (genome-wide p-value < 0. 001) at 30 minutes post induction, the time at which heterogeneity markedly differed between the BY and RM strains (Fig 7B and 7C). The locus was also significant at times 20 min (p < 0. 005) and 40 min (p < 0. 005) post induction. Visualizing the distributions of single-cell expression levels at 30 minutes revealed that the RM and BY genotypes at this locus corresponded to high and low cell-cell heterogeneity, respectively (Fig 7D and 7E). Thus, this locus explains, at least in part, the different levels of heterogeneity observed between the parental strains. It should therefore also be detected as a varQTL or cvQTL. This was indeed the case: the LOD score linking the locus to the variance of expression was 4. 5 and reached statistical significance (P = 0. 005). Importantly, the scPTL was not a QTL: the locus genotype did not correlate with the mean level of expression of the population of cells (LOD score < 2. 8). When surveying the genomic annotations of the locus [40], we realized that it contained no obvious candidate gene that would explain an effect on the heterogeneity of the response (such as genes known to participate to the transcriptional response). One potentially causal gene was DOT6, which encodes a poorly characterized transcription factor that was shown to shuttle periodically between the cytoplasm and nucleus of the cells in standard growth conditions [41]. Given that i) the shuttling frequency of such factors can sometimes drive the response to environmental changes and ii) numerous non-synonymous BY/RM genetic polymorphisms were present in the gene, we constructed an allele-replacement strain for DOT6 and tested if the gene was responsible for the scPTL linkage. This was not the case. Strains BY and BY-DOT6RM (isogenic to BY except for the DOT6 gene which was replaced by the RM allele) displayed very similar transcriptional responses at intermediate times of induction (S1 Fig). Fine-mapping of the locus and a systematic gene-by-gene analysis are now needed to precisely identify the polymorphisms involved. By highlighting a novel genetic locus modulating cell-cell variability of the transcriptional response to galactose, our results show that scPTL scanning can provide new knowledge on the fine structure of a well-studied system. When considering macroscopic phenotypes, it is important to distinguish the situations where scPTL mapping is biologically relevant from those where it is not. The determinants of human height, for example, act via countless cells, of multiple types, and over a very long period of time (~ 16 years). In such cases, the macroscopic trait results from multiple effects that are cumulated and considering the probabilistic individual contribution of specific cells is inappropriate. Similarly, many tissular traits heavily rely on communications between cells and probabilistic changes in a few may not affect the collective output of the cell population. In contrast, a number of macroscopic traits can be affected by particular events happening in rare cells or at a very precise time (see below). In these cases, studying the probabilities of a biological outcome in the relevant cells or of a molecular event within the critical time interval can provide invaluable information on the emergence of the macroscopic phenotype, and scPTL mapping then becomes relevant. A striking example of such traits is cancer. Genetic predisposition is conferred by variants affecting somatic mutation rates and these loci are special cases of scPTL: the cellular trait they modify is the amount of de novo mutations in the cell' s daughter. These variants have classically been identified by genetic linkage of the macroscopic trait (disease frequency in families and cohorts), and their role on the maintenance of DNA integrity was deduced afterwards by molecular characterizations. For a review on the genetics of cancer syndrome predisposition, see [42,43]. scPTL mapping is also relevant to the non-genetic heterogeneity of cancer cells which was shown to be associated with tumour progression [7,8] and treatment efficiency [9–11]. Genetic loci changing the fraction of problematic cells are likely modulators of the prognosis. If the functional properties (expression level, phosphorylation status, subcellular localization) of a key molecular player, such as a critical tumor-suppressor gene, can be monitored in numerous individual cells, then scPTL mapping, as presented here, may help identify genetic factors that modulate the activity of this gene in a probabilistic manner. Once identified, the association of these loci with the macroscopic phenotype can then be tested directly, avoiding at least partly the statistical challenges of whole-genome scans. To illustrate this, we considered an idealized case where three scales are bridged: at the molecular level, a scPTL affects the expression of a protein X (same regulation as in Fig 4); at the cellular level, cells have higher probability to divide if their level of X is low (Fig 8A); and at the whole-organism level, disease appears if too many cells are present. Using a stochastic model of this scenario, we simulated a cohort of individuals and recorded the state and number of cells in every individual over time (Fig 8B, see methods for details). Disease appeared in all individuals, between age 22 and 29. Using the data at age 23, we compared the power of GWAS and scPTL mapping. For GWAS, the trait of individuals was whether they had declared the disease or not. For scPTL mapping, the trait was the expression level of X in 10,000 of their cells. As expected by the moderate effect on disease frequency, GWAS failed to detect the locus (Fig 8C). In contrast, scPTL detection was highly significant from the same cohort of individuals (Fig 8D). Importantly, although not significant genome-wide, the GWAS score at the locus had a nominal p-value lower than 0. 01 (Fig 8C). The locus would therefore be considered significant if it had been the only one tested. This illustrates the added value of scPTL mapping: while keeping cohort size constant, it can highlight candidate loci of the genome that can then be tested individually for association to the disease. This power clearly results from i) additional traits (cellular ones) that are included before scanning the genome and ii) relaxation of multiple-testing correction when testing association to disease. Note that other system genetics methods, such as expression QTL (eQTL) mapping, improve power in a similar way: they highlight relevant candidates via the addition of intra-individual traits (molecular ones) [44]. Note also that recruiting large cohorts remains important: Methods detecting scPTL and eQTL can improve genetic mapping but their detection power remain strictly dependent on the number of individuals available in the study. In real studies, external knowledge is needed on the link between the cellular trait and the disease: what single-cell trait should be measured? Can it be measured in a sufficiently large number of cells? If a reporter system of de novo mutations, for example based on the intracellular distribution of a fluorescently tagged repair protein [45,46], can be introduced in a relevant and large population of cells, then the high number of cell measurements may allow to detect loci that modify even slightly the mutation rate. For non-genetic features of problematic cells, choice of the trait can be driven by investigations at the molecular level, such as stochastic profiling [47], and at the cellular level, such as recording the response of cell populations to treatment or differentiation signals [9,10]. For example, the distribution of the biomarker JARID1B (a histone demethylase) in populations of melanoma cells is indicative of an intra-clonal heterogeneity that is important for tumour progression [7], biomarkers CD24 and CD133 can distinguish rare cells that persist anti-cancer drug treatments [10] and multiplexed markers of signalling response can reveal patterns of population heterogeneity that predict drug sensitivity [48]. When relevant markers are not known, a possibility is to screen for them using stochastic profiling [47]. This method interrogates the transcriptomic variability between pools of few cells in order to identify transcripts displaying elevated cell-to-cell variability in specific biological contexts. It allowed the discovery of two molecular states of extracellular matrix-attached cells that can be distinguished by the jun D proto-oncogene and markers of TGF-β signalling [8]. Such markers of isogenic cellular subtypes may allow the development of scPTL mapping in humans. An important statistical requirement to identify scPTL is the abundance of cells on which the probabilistic trait is quantified. For human studies, peripheral blood offers access to many cells but, unfortunately, many internal organs do not. This requirement also implies using technologies where the throughput of quantitative acquisitions is high. This is the case for flow-cytometry and, although at higher costs, for high-content image analysis [14,15] and digital microfluidics [17,18]. For these practical reasons, it is possible that mouse immunological studies will help making progress in mammalian scPTL mapping. For example, the work initiated by Prince et al. [49] describing pre- and post-infection flow-cytometry profiles of F2 offsprings from different mouse strains may provide an interesting pilot framework. The interest of scPTL mapping is not restricted to cancer biology. Developmental processes and cellular differentiation are also vulnerable to mis-regulations happening in few cells or during short time intervals. Their macroscopic outcome can therefore be affected by probabilistic events at the cellular scale. For example, stochastic variation in the expression of the stem cell marker Sca-1 is associated with different cellular fates in mouse hematopoietic lineages [50], suggesting that genetic factors changing this stochastic variation may impact blood composition. Similarly, embryonic stem cells co-exist in at least two distinct molecular states that are sensitive to epigenetic and reprogramming factors [51]. Genetic variants modulating these factors may change the statistical partitioning of these states. Two observations made on flies remarkably support the existence of natural genetic factors altering developmental processes in a probabilistic manner. The first one is the fact that high levels of fluctuating asymmetry can be fixed in a wild population of D. melanogaster under artificial selection [52]. The second one comes from a comparative study of Drosophila species [53]. Embryos of D. santomea and D. yakuba display high inter-individual variability of expression of the signal transducer pMad at the onset of gastrulation, as compared to D. melanogaster embryos. This increased variability was attributed to a reduced activity of the homeobox gene zerknüllt thirty minutes before this stage. Very interestingly, it is accompanied by phenotypic variability (inter-individual variance of the number of amnioserosa cells) in D. santomea but not in D. yakuba. These and other examples [54] illustrate how developmental variability and phenotypic noise can evolve in natural populations. Applying scPTL mapping may allow to dissect the genetic factors responsible for this evolution. Our new method based on the Kantorovich distance is not the only one by which scPTL can be identified. Applying classical QTL mapping to summary statistics of the cellular traits can also be efficient. We emphasize that the two approaches are complementary. For example, our method missed to detect linkage for 9 yeast morphological traits for which cvQTL scans were successful, but it detected several significant scPTL that were missed by the QTL-based approach (Figs 5 and 6). Second, we observed that scPTL detection was often efficient when the mean value of cellular trait differed among genotypic categories. As shown on Fig 5B, traits successfully mapped tended to display high heritability of the mean. Thus, after a scPTL is detected, it is necessary to examine the effect on the trait distributions and to determine if it is a QTL or not. Third, alternative ways of mapping scPTL are open and may prove more appropriate in some contexts. For example, if a cellular trait becomes preoccupying when it exceeds a certain threshold value, then the fraction of cells above this threshold can be used as a macro-trait to be mapped by QTL analysis. This way, the focus is made on the relevant aspect of the cellular trait, avoiding variation in other parts of the distribution. We therefore recommend conducting Kantorovich-based scPTL mapping in addition to classical methods and not as a replacement strategy. While the principle of genotype-phenotype genetic linkage dates back to several decades ago, the statistical methods that test for linkage are still being improved, especially regarding multi-loci interactions or population structure corrections [55,56]. The present study provides a priming of a generic scPTL mapping approach (exploiting thousands of single-cell trait values) and demonstrates its feasibility and potential (new loci were detected). Since it is new, we anticipate that it will also evolve in the future. It is currently based on three steps: (i) computing pairwise distances between individuals by using the Kantorovich metric, (ii) using the resulting distance matrix to construct a relevant phenotypic space and (iii) testing for genetic linkage by LDA. A number of methodological considerations can be made in anticipation to future developments and applications. Estimating the proportion of variance explained by scPTL is not straightforward: the' captured variance' as quantified by the eigenvalues of the LDA is not the same as the' explained variance' which must be re-computed by regression; and if linearity of the data is questionable, the method remains a useful tool if it detects scPTL but interpreting variance proportions need justifications. A phenotypic space can be constructed by alternative ways that do not require the Kantorovich metric. For example, we considered representing individuals in a" space of moments", where the coordinate of every individual on the i-th axis was the i-th moment of the cellular trait distribution associated to this individual. We applied this to the yeast morphological data and we searched for genetic linkages by linear discriminant analysis as described above. This approach detected many significant scPTL but we encountered a difficulty that was avoided by our Kantorovich-metric based method. When searching for significant linear discrimination, the dimensionality of the phenotypic space is important. At high dimensionality, discriminant axes are more likely to be found. This improves detection in the actual data but at the expense of increasing the degrees of freedom and therefore the false positive hits estimated from the permuted data. In a" space of moments", the properties of the single-cell trait distributions are very important because they define which axis (moments) are relevant to separate individuals. Keeping the 4-th axis may be crucial even if all individuals have very similar first, second or third moments. Choosing the appropriate dimension for LDA is then arbitrary and it becomes difficult to keep a good detection power while still controlling the FDR. In fact, applying QTL mapping on the 3rd and 4th moments of all traits was fruitless because the FDR could not be controlled at the genome-by-phenome scale. This issue is avoided in the case of Kantorovich distances because multi-dimensional scaling can be applied without normalization and the axes of the phenotypic space are ranked by descending order of their contribution to the inter-individual differences. The 4-th axis, for example, contributes less than the first three axes to the separation of individuals in the space. If keeping the 4-th dimension prior to LDA is beneficial for linkage, then keeping the first three axes is also highly relevant, and this is true regardless of the properties of the single-cell trait distributions. We found this very useful: our algorithm adds dimensions one by one and evaluates the benefit of each increase (see methods). There are at least three lines along which our method may be further improved. First, LDA is only appropriate if genotypic categories can be distinguished along linear axis. If individuals in the phenotypic space are separated in non-linear patterns, other methods such as those based on kernel functions [57] may be more appropriate. Second, we propose to compute confidence intervals of scPTL position by bootstrap, following a method sometimes applied to QTL positions [58]. As expected, resampling not only affected scPTL position but also the optimal dimensionality retained (S2A Fig). A deeper investigation of the simultaneous variation of these two outputs could help improve the precision of mapping. And third, single-cell data acquisitions often generate multiple trait values for each individual cells. This is the case for morphological profiling as in the dataset we used here, but also for gene expression [59] or parameters describing the micro-environment of the cells [60]. It would therefore be interesting to search for scPTL affecting multiple cellular traits simultaneously instead of treating cellular traits one by one. A multidimensional analysis could be performed in order to extract a set of informative meta-traits, such as principal components or representative medoids and scPTL of these meta-traits could be searched using our method. This dimension-reduction approach would benefit from the redundant information available from correlated traits (e. g. the perimeter of a cell and its area are two measurements of its size), but the biological interpretation of a probabilistic effect on a meta-trait may not be straightforward. Alternatively, one might want to identify scPTL affecting the joint probability distribution of multiple cellular traits. In this case, a natural extension of our method would be to compute Kantorovich distances between multivariate distributions. However, the Kantorovich metric cannot be easily computed for more than two marginals (i. e. cellular traits in our case). In fact, its existence as a unique solution to the multi-dimensional transportation problem was itself a subject of research [61]. A possible alternative could be to compute a Euclidean distance in the" space of moments" mentioned above and then apply multi-dimensional scaling. Furthermore, although our study was focused on probability density functions, steps (ii), constructing the phenotypic space, and (iii), testing for genetic linkage, could in principle be applied to other types of functions, provided that a relevant metric estimating the dissimilarity between such functions exists. This could be interesting in the case of function-valued traits, such as speech sound or other time-series functions. The evolution of these functions is being studied using phylogenetic methods that present challenging statistical issues [62,63]. Extending our approach to such functions may open the possibility to study them from a (complementary) quantitative genetics angle. Finally, we can anticipate that gene-gene and gene-environment interactions also shape the probability density function of cellular traits. Our results on the activation of the yeast galactose network remarkably illustrate this: the effect of the scPTL on chromosome V is apparent only transiently, and in response to a change of environmental conditions. It is tempting to extrapolate that signaling pathways in plants and animals may be affected by scPTL that act at various times and steps along molecular cascades. In conclusion, our study provides a novel method that can detect genetic loci with probabilistic effects on single-cell phenotypes, with no prior assumption on their mode of action. By exploiting the power of single-cell technologies, this approach has the potential to detect small-effect genetic variants that may underlie incomplete trait penetrance at the multicellular scale. Single-cell gene activity was modeled by a stochastic variable X that represented the number of proteins in one cell at a given time. Under the model, the dynamics of X is controlled by two processes: (1) protein production with rate α and (2) protein degradation with rate β. We assume that the gene is positively auto-regulated by a 4-mer complex, meaning that α is an increasing function of X with a typical Hill-like shape α = α0+α1 (XK) 41+ (XK) 4 with α0 the leaky production rate in absence of X 4-mers at the promoter, α1 the production rate in presence of 4-mers, and K the dissociation constant of the 4-mer. We set β = 1, which corresponds to scaling time units. The dynamics of the mean value of X in a population of isogenic cells follows the equation shown in Fig 4A. To obtain the probability distribution of X, we performed exact stochastic simulations of the chemical system defined by the two reactions rate α and β, using the Stochastic Simulation Algorithm [64]. To generate two groups of individuals, we assumed that the set of parameters (α0, α1, K) was controlled by one locus that could exist in two alleles (A and B) with mean values (μ0A/B, μ1A/B, μKA/B) and, for simplicity, that the individuals were haploids. To account for sources of inter-individual variability within genotypic groups, the values of the parameters for one individual were drawn from normal distributions of mean values μ0A/B, μ1A/B and μKA/B and of standard deviations ημ0A/B, ημ1A/B and ημKA/B where η represented the strength of inter-individual variability. η was assumed to be the same for A and B alleles. Values were: μ0A = 6. 3, μ0B = 0. 1, μ1A = 12, μ1B = 10, μKA = 10 and μKB = 1. 6. All statistical analysis were done using R (version 3. 1. 2) [65]. The data from Nogami et al. [22] consisted of 220 traits, acquired on >200 cells per sample. Note that most traits are related to one of three division stages. Each trait was therefore measured on a subset of cells of the sample (less than 200). There were nine samples of the BY strain, nine of the RM strain, and three of each of 59 segregants of the BY x RM cross. For each trait, we computed the genetic heritabilities of the mean and CV as follows. The mean and CV of the cellular trait in each sample were computed, leading to two scalar values per sample that we call macro-traits hereafter (to distinguish them from the single-cell values). The broad-sense genetic heritability of each macro-trait was H2 = (varT − varE) / varT, with varT and varE being the total and environmental variance, respectively. For Fig 2B, we estimated varT by randomly choosing one of the three replicate sample of each segregant and computing the variance across these 59 values. This was repeated 100 times and the estimates were averaged. Our estimate of varE was the pooled variance of varBY, varRM, varSeg1, varSeg2, …, varSeg59 which were the between-replicates variance of each strain. Confidence intervals on H2 values were computed by bootstrapping the strains. For the filtering step prior to linkage, H2 was computed slightly differently in order to be consistent across mapping methods (see below). We first normalized the distributions as densities (division of all bin counts by half the total number of cells). Following [27], we then computed the Kantorovich distance between two distributions f1 and f2 as the area under the absolute value of the cumulative sum of the difference between the two distributions: KD (f1, f2) = ∫−∞+∞|∫−∞xf1 (t) −f2 (t) dt|dx Multi-dimensional scaling of the resulting distance matrix was then performed using the R function cmdscale () from the stats package. The number of dimensions retained (ndim) was the number of eigenvalues exceeding the expected value under the hypothesis of no structure in the data (i. e. mean of all eigenvalues, Kaiser criterion). We computed the heritability of each yeast morphological trait in this multidimensional space. This was done as above for one dimension, by computing the total variance of the data, and estimating the environmental variance from the replicated experiments made on the parental strains. For 147 traits, heritability was greater than 0. 5 and scPTL were searched. Details on how these steps were implemented in R are described in S1 Methods, and the code is available in the open source ptlmapper R package (https: //github. com/fchuffar/ptlmapper). The yeast genotypes we used were from Smith and Kruglyak [66]. For the morphological traits, we pooled triplicates together in order to increase the number of cells per sample. The data then corresponded to 220 traits, measured on >600 cells per sample, with 3 BY samples, 3 RM samples, and 1 sample per segregant. We scanned the genetic map with two methods. First, we considered the coordinates of each segregant on the first axis of the multi-dimensional scaling, and we considered this coordinate as a quantitative trait that we used for interval mapping using R/qtl [67]. Secondly, we applied a linear discrimination analysis (LDA) on the phenotypes data, using the genotype at every marker as the discriminating factor. An important issue in this step is the multidimensionality of the data: axis 2,3 and more may contain useful information to discriminate genotypic groups, but if too many dimensions are retained, a highly-discriminant axis may be found by chance only. To deal with this issue, we evaluated the output of LDA at all dimensions d ranging from 2 to ndim. For each value of d, we applied LDA at every marker position and we recorded the Wilks' lambda statistics: Λ = ∏j = 1d 11+λj where λj was the j-th eigenvalue of the discriminant analysis. Low values of this statistics allow to reject the null hypothesis of no discrimination by the factor of interest [68] which, in our case, is the genotype. We defined a linkage score (W score) as: W = −Log10 (P) where P is the p-value of the Wilk' s test (deviation of Λ from the F-statistics with relevant degrees of freedom). Note that P is not interpreted directly as a significance value for linkage (see the permutation test below). We then quantified how much the best marker position was distinguished from the rest of the genome by computing a Z-score: Z = Wbest − Wσw where Wbest, <W> and σW were the highest, the mean and the standard deviation of all W scores found on the genome, respectively. Finally, we chose the dimension that maximized this Z-score (i. e. dimension where the linkage peak had highest contrast). Very importantly, the same degrees of freedom (exploration of the results at various dimensionalities) were allowed when applying the permutation test of significance (see below). The distribution of the dimensionalities retained for the morphological traits is shown in S2B Fig. Additional details are provided in S1 Methods and the code is available in the open source ptlmapper R package (https: //github. com/fchuffar/ptlmapper). QTL-based mapping was performed as follows. A quantitative trait was considered at the cell-population level. This macro-trait was either the mean (for QTL), the coefficient of variation (standard deviation divided by mean, for cvQTL) or the variance (for varQTL) of the cellular trait in the population of cells. For the yeast morphology data, we selected the traits with H2 > 0. 5 prior to linkage. To do so, we re-computed H2 values in a way that was consistent with the heritability calculation of the phenotypic space prior to scPTL mapping, where replicates of segregants were pooled together before analysis of inter-strain variation (see above). In this case, only 3 replicates of BY and 3 replicates of RM are then available to estimate the environmental variance. Therefore, we estimated varT as the variance of the 59 macro-trait values of the segregants and varE as (varBY + varRM) / 2, with varBY (resp. varRM) being the variance of the three macro-trait values of the BY (resp. RM) strain. We then scanned the genome using the scanone function from r/qtl [67] with a single QTL model and the multiple imputation method [69]. Our code implementing the calls to r/qtl is available in the open source ptlmapper R package (https: //github. com/fchuffar/ptlmapper). We first explain the case where a single trait is studied. When the trait was mapped using R/qtl, significance was assessed by the permutation test implemented in function scanone () of the package [67]. For scPTL, we implemented our own permutation test as follows. The significance of an scPTL is the type one error when rejecting the following null hypothesis:" there is no marker at which the genotype of individuals discriminates their location in the phenotypic space", where one' individual' refers to one population of isogenic cells, and where the' phenotypic space' is the multi-dimensional space built above by computing Kantorovich distances and applying multi-dimensional scaling. The relevant permutation is therefore to randomly re-assign the phenotypic positions to the individuals before scanning genetic markers for discrimination. We did this 1,000 times. Each time, LDA was applied at dimensions 2 to ndim, the dimension showing the best contrast (high Z score) was retained, and the highest W score obtained at this dimension was recorded. The empirical threshold corresponding to genome-wide error rates of 0. 1%, 1% and 5% were the 99. 9th, 99th and 95th percentiles of the 1,000 values produced by the permutations, respectively. These thresholds are typically those employed in whole-genome scans for a single trait. We now explain the case of the morphological study, where multiple traits (220) were considered. This case is similar to system genetics studies, where the FDR must be controlled. Keeping it below 10% ensures that 9 out of 10 results are true positives, which is often considered as acceptable. Four different methods were used. For three of them, single-cell trait values were resumed to a scalar macro-trait and QTL was searched. The three methods differed by the choice of this macro-trait, which was either the mean or the coefficient of variation of single-cell traits, or the coordinate of individuals on the first axis of the phenotypic space. For each of the three methods, morphological traits with less than 50% genetic heritability (see above) were not considered further, and QTL was searched for the remaining Ntraits traits only. For each of these traits, LOD scores were computed on the genome by interval mapping using the macro-trait value as the quantitative phenotype of interest. Significance was assessed by random re-assignment of the macro-trait values to the individuals (yeast segregants). We did 1,000 such permutations. For each one, the genome was scanned as above and the highest LOD score on the genome was retained. This generated a 1,000 x Ntraits matrix Mperm of the hits expected by chance. At a LOD threshold L, the FDR was computed as: FDR = NFalseL / NActualL where NActualL was the number of linkages obtained from the actual dataset at LOD > L, and NFalseL was the expected number of false positives at LOD > L, which was estimated by the fraction of elements of Mperm exceeding L. The fourth method considered all coordinates of the individuals in the phenotypic space. At this step, for each morphological trait, a phenotypic space of ndim dimensions had been built as explained above by computing Kantorovich distances and applying multi-dimensional scaling. Let P1, P2 and PS be the phenotypic matrices of parent 1 (strain BY), parent 2 (strain RM) and segregants, respectively, with rows being the samples (replicates for P1 and P2, and segregants for PS) and columns being the ndim coordinates of each sample in the phenotypic space. These matrices had dimensions 3 x ndim for P1 and P2 and 59 x ndim for PS. Genetic heritability was computed as H2 = (varT—varE) / varT, where the total variance varT was the variance of the samples in PS, and where the environmental variance varE was estimated as (varP1 + varP2) / 2, with varP1 (resp. varP2) being the variance of the samples in P1 (resp. P2). Morphological traits showing H2 < 0. 5 were discarded, and scPTL mapping was applied to the remaining Ntraits traits as described above (choice of dimensionality with highest contrast and recording of the best W score obtained on the genome at this dimensionality). Significance of W scores was assessed as described above for the LOD scores, by performing 1,000 permutations and determining the FDR associated to various thresholds of W scores. For each cell, the probability to divide depended on the concentration of gene product X according to the following Hill-like function (Fig 8A): P (X) = β01+ (Xθ) n+ β∞ with β0 = 0. 2, ϑ = 2. 5, β∞ = 0. 05 and n = 2. 5. The regulation of X was governed by the same model as above, with η = 0. 16. For each individual, parameters alpha0, alpha1 and K were drawn from the same normal distributions as above, where mean and variance depended on the genotype (A or B). At age 0, a population of 1,000 cells was initiated with X = 5. This population was then evolved by Stochastic Simulation Algorithm [64], with a constant rate of cell death of 0. 0001 until the age of 30. The python code implementing this simulation is provided in S2 Methods. The yeast strains and oligonucleotides used in this study are listed in S2 Table. To construct the Gal-GFP reporter, we first removed the MET17 promoter of plasmid pGY8 [19] by digestion with restriction enzymes BspEI and SpeI followed by Klenow fill-in and religation. This generated plasmid pGY10. The GAL1 promoter fragment was digested (BglII-BamHI) from pFA6a-His3MX6-PGAL1 [70] and cloned in the BamHI site of pGY10. A small artificial open reading frame upstream GFP was then removed by digestion with EcoRV and BamHI, Klenow fill-in end blunting and religation. This generated plasmid pGY37, carrying a PGAL1-yEGFP-NatMX cassette that could be integrated at the HIS3 genomic locus. Plasmid pGY37 was linearized at NheI and integrated at the HIS3 locus of strain BY4716 (isogenic to S288c), YEF1946 (a non- clumpy derivative of RM11-1a) and in 61 F1 non-clumpy segregants from BY471xRM11-1a described in [22] to generate strains GY221, GY225, and the S288c x RM11-1a HIS3: PGAL1-yEGFP-NatMX: HIS3 set, respectively. In parallel, we also constructed a GAL1-GFPPEST reporter coding for a destabilized fluorescent protein [71]. We derived it from pGY334, where GFPPEST was under the control of the PGK promoter. pGY334 was constructed in several steps. The PGK promoter was PCR-amplified from pJL49 (gift from Jean-Luc Parrou) using primers 1A23 and 1A24, digested by BamHI and cloned into the BamHI site of pGY10. The resulting plasmid was digested with EcoRV and XbaI, subjected to Klenow fill-in end blunting and religated, generating plasmid pGY13 carrying a HIS3: PPGK-yEGFP-NatMX: HIS3 cassette. The lox-CEN/ARS-lox sequence from pALREP [20] was amplified by PCR using primers 1I27 and 1I28 and cloned by homologous recombination into pGY13, generating plasmid pGY252. The GFPPEST sequence was PCR-amplified from pSVA18 [71] using primers 1I92 and 1I93 and cloned in vivo into pGY252 (digested by MfeI and DraIII), leading to pGY334. The GAL1 promoter fragment was amplified by PCR from pGY37 using primers 1J33 and 1I42 and cloned into plasmid pGY334 by recombination at homologous sequences flanking the BamHI site of the plasmid. The CEN/ARS cassette of the resulting plasmid was excised by transient expression of the Cre recombinase in bacteria [20], generating the final integrative plasmid pGY338 carrying the HIS3: PGAL1-GFPPEST-NatMX: HIS3 cassette. pGY338 was linearized by NheI and integrated at the HIS3 locus of BY4724 (isogenic to S288c) and GY1561 to create GY1566 and GY1567 strains, respectively. Strain GY1561 is a non-clumpy derivative of RM11-1a where the KanMX4 cassette was removed. It was obtained by first transforming RM11-1a with an amplicon from plasmid pUG73 [72] obtained with primers 1E75 and 1E76 and selecting a G418-sensitive and LEU+ transformant (GY739) which was then transformed with pSH47 [73] for expression of the CRE recombinase. After an episode of galactose induction, a LEU- derivative was chosen and cultured in non-selective medium (URA+) for loss of pSH47, leading to strain GY744, which was then crossed with GY689 [74] to generate GY1561. Liquid cultures in synthetic medium with 2% raffinose were inoculated with a single colony and incubated overnight, then diluted to OD600 = 0. 1 (synthetic medium, 2% raffinose) and grown for 3 to 6 hours. Cells were then resuspended in synthetic medium with 2% raffinose and 0. 5% galactose and grown for the desired time (0,10,20,30,40,60,80,100,130,160,205 and 250 minutes). Cells were then washed with PBS1X, incubated for 8 min in 2% paraformaldehyde (PFA) at room temperature, followed by 12 min of incubation in PBS supplemented with Glycine 0. 1M at room temperature and finally resuspended in PBS. They were then analyzed on a FACSCalibur (BD Biosciences) flow cytometer to record 10,000 cells per sample. Flow cytometry data was analysed using the flowCore package from Bioconductor [75]. Cells of homogeneous size were dynamically gated and normalized as follows: (i) removal of events with saturated signals (FSC, SSC or FL1 ≥ 1023 or ≤ 0), (ii) correction of FL1 values by subtracting the mean (FL1) observed on the same strain at t = 0, (iii) computation of a density kernel of FSC, SSC values to define a perimeter of peak density containing 60% of events, (iv) cell gating using this perimeter and (v) removal of samples containing less than 3,000 cells at the end of the procedure. The GFP expression values were the corrected FL1 signal of the retained cells. The DOT6RM allele was amplified by PCR from genomic DNA of the RM strain using primers 1K87 and 1K88. It was then cloned into plasmid pALREP [20] by homologous recombination at sequences flanking the HpaI site of the plasmid. The CEN/ARS cassette of the resulting plasmid was excised by transient expression of the Cre recombinase in bacteria, as previously described [20], generating plasmid pGY389, which was linearized at EcoRI (a unique site within the DOT6 gene) and integrated in strain GY1566 (isogenic to BY, and carrying the HIS3: PGAL1-GFPPEST: HIS3 cassette). The pop-in pop-out strategy was applied as previously described [20] and four independent transformants were selected (GY1604, GY1605, GY1606 and GY1607) where PCR and sequencing validated the replacement of the DOT6 allele. The yeast morphological data corresponds to the experiments described in [22]. For the present study, raw images were re-analyzed using CalMorph 1. 0. The single-cell values and genotypes used are provided in S1 Dataset of this article. The flow cytometry data corresponding to yeast galactose response is made available from http: //flowrepository. org under accession number FR-FCM-ZZPA. The simulated data of Fig 4 is available as an R package (ptldata) from https: //github. com/fchuffar/ptldata. The scPTL mapping method is made available as an open source R package (ptlmapper) which can be downloaded from https: //github. com/fchuffar/ptlmapper. A tutorial of this package explains how to run the analysis on the simulated dataset. | Title: Exploiting Single-Cell Quantitative Data to Map Genetic Variants Having Probabilistic Effects Summary: Genetic association studies are usually conducted on phenotypes measured at the scale of whole tissues or individuals, and not at the scale of individual cells. However, some common traits, such as cancer, can result from a minority of cells that adopted a special behavior. From one individual to another, DNA variants can modify the frequency of such cellular behaviors. The body of one of the individuals then harbours more misbehaving cells and is therefore predisposed to a macroscopic phenotypic change, such as disease. Such genetic effects are probabilistic, they contribute little to trait variation at the macroscopic level and therefore largely escape detection in classical studies. We have developed a novel statistical method that uses single-cell measurements to detect variants of the genome that have non-deterministic effects on cellular traits. The approach is based on a comparison of distributions of single-cell traits. We applied it to colonies of yeast cells and showed that it can detect mutations that change cellular morphology or molecular regulations in a probabilistic manner. This opens the way to study multicellular organisms from a novel angle, by exploiting single-cell technologies to detect genetic variants that predispose to certain diseases or common traits. | 15,689 | 279 | lay_plos | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Virtual Screening for Colorectal Cancer Act of 2010''. SEC. 2. COVERING SCREENING COMPUTED TOMOGRAPHY COLONOGRAPHY AS A COLORECTAL CANCER SCREENING TEST UNDER MEDICARE.. (a) In General.--Section 1861(pp)(1) of the Social Security Act (42 U.S.C. 1395x(pp)(1)) is amended-- (1) by redesignating subparagraph (D) as subparagraph (E); and (2) by inserting after subparagraph (C) the following new subparagraph: ``(D) Screening computed tomography colonography.''. (b) Frequency Limits and Payment.--Section 1834(d) of such Act (42 U.S.C. 1395m(d)) is amended by adding at the end the following new paragraph: ``(4) Screening computed tomography colonography.-- ``(A) Fee schedule.--With respect to a colorectal cancer screening test consisting of screening computed tomography colonography, subject to subparagraph (B), payment under section 1848 shall be consistent with payment under such section for similar or related services. ``(B) Payment limit.--In the case of screening computed tomography colonography, payment under this part shall not exceed such amount as the Secretary specifies. ``(C) Facility payment limit.-- ``(i) Limitation on coinsurance.-- Notwithstanding any other provision of this title, in the case of an individual who receives screening computed tomography colonography-- ``(I) in computing the amount of any applicable coinsurance, the computation of such coinsurance shall be based upon the fee schedule under which payment is made for the services; and ``(II) the amount of such coinsurance shall not exceed 25 percent of the payment amount under the fee schedule described in subparagraph (A). ``(D) Frequency limit.--No payment may be made under this part for a colorectal cancer screening test consisting of a screening computed tomography colonography-- ``(i) if the individual is under 50 years of age; or ``(ii)(I) in the case of individuals at high risk for colorectal cancer, if the procedure is performed within the 23 months after a previous screening computed tomography colonography or a previous screening colonoscopy; or ``(II) in the case of an individual who is not at high risk for colorectal cancer, if the procedure is performed within the 119 months after a previous screening colonoscopy or within the 59 months after a previous screening flexible sigmoidoscopy or a previous screening computed tomography colonography.''. (c) Conforming Frequency Limits for Other Colorectal Cancer Screening Tests.-- (1) Screening flexible sigmoidoscopy.--Paragraph (2)(E)(ii) of section 1834(d) of the Social Security Act (42 U.S.C. 1395m(d)) is amended by inserting ``or screening computed tomography colonography'' after ``previous screening flexible sigmoidoscopy''. (2) Screening colonoscopy.--Paragraph (3)(E) of such section is amended-- (A) by inserting ``or screening computed tomography colonography'' after ``23 months after a previous screening colonoscopy''; and (B) by inserting ``or screening computed tomography colonography'' after ``screening flexible sigmoidoscopy''. (d) Effective Date.--The amendments made by this section shall apply to items and services furnished on or after January 1, 2011. SEC. 3. EXEMPTION OF SCREENING COMPUTED TOMOGRAPHY COLONOGRAPHY FROM SPECIAL RULE ON PAYMENT FOR IMAGING SERVICES. (a) In General.--Section 1848(b)(4)(B) of the Social Security Act (42 U.S.C. 1395w-4(b)(4)(B)) is amended by inserting ``and screening computed tomography colonography'' after ``diagnostic and screening mammography''. (b) Effective Date.--The amendment made by subsection (a) shall apply to items and services furnished on or after January 1, 2011. SEC. 4. REPORT ON THE EFFECTIVENESS OF SCREENING CTC IN IMPROVING OVERALL CRC COMPLIANCE. (a) GAO Study and Reports on Effectiveness of Screening Computed Tomography Colonograpy in Improving Overall Colorectal Cancer Compliance.-- (1) Study.-- (A) In general.--The Comptroller General of the United States (in this subsection referred to as the ``Comptroller General'') shall conduct a study, by colorectal cancer screening procedure type, on-- (i) the effect of the addition of the screening computed tomography colonography (in this subsection referred to as ``screening CTC'') benefit under section 1861(pp)(1)(D) of the Social Security Act, as added by section 2(a); (ii) the effect of the addition of such benefit as part of an overall set of colorectal cancer screening (in this subsection referred to as the ``CRC screening'') made available to relevant Medicare population, including individuals over 50 years of age and over 75 years of age; and (iii) any other relevant questions involving access to, beneficiary preference of, and the value of, screening CTC for Medicare beneficiaries. (B) Issues.--The study conducted under subparagraph (A) shall examine the following: (i) The impact of screening CTC on the change in CRC screening compliance of Medicare beneficiaries. (ii) The various utilization rates for the each available CRC screening option before and after the availability of screening CTC, including-- (I) by initial CRC screening performed as a Medicare beneficiary, including the beneficiary age when initial screening was performed; and (II) by follow-on screening performed, whereby the analysis demonstrates to what extent screening CTC was used as a substitute for a previous screening procedure. (iii) The referral rate of screening CTC to follow-on optical colonoscopy. (iv) An analysis of beneficiary preference to the various CRC screening options. (v) Access to screening CTC by Medicare beneficiaries, especially in rural areas or underserved populations, before and after implementation of such screening benefit. (vi) Such other issues as the Comptroller General determines appropriate. (2) Reports.-- (A) Preliminary report.--Not later than March 1, 2015, the Comptroller General shall submit a preliminary report to Congress on the study conducted under paragraph (1). (B) Final report.--Not later than March 1, 2016, the Comptroller General shall submit a final report to Congress on the study conducted under paragraph (1), together with recommendations for such legislation and administrative action as the Comptroller General determines appropriate. | Title: To amend title XVIII of the Social Security Act to cover screening computed tomography colonography as a colorectal cancer screening test under the Medicare Program Summary: Virtual Screening for Colorectal Cancer Act of 2010 - Amends title XVIII (Medicare) of the Social Security Act to: (1) provide Medicare coverage for screening computed tomography colonography (CTC) as a colorectal cancer (CRC) screening test; and (2) exclude screening CTC from the meaning of "imaging services" for which there is a special rule regarding outpatient services department (OPD) fee schedule payments. Directs the Comptroller General to study, by CRC screening procedure type, and report to Congress on: (1) the effect of the addition of the screening CTC benefit under this Act; (2) the effect of the addition of such benefit as part of an overall set of CRC screening made available to relevant Medicare population, including individuals over age 50 and over age 75; and (3) any other relevant questions involving access to, beneficiary preference of, and value of, screening CTC for Medicare beneficiaries. | 1,633 | 240 | billsum | en |
Summarize: CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to international patent application PCT/JP2005/021322 filed on Nov. 15, 2005, and Japanese patent applications 2005-118919 filed on Mar. 21, 2005 and 2005-303644 filed on Sep. 16, 2005. TECHNICAL FIELD [0002] This invention relates to a siphon unit for rapidly discharging liquid. BACKGROUND [0003] In an aquarium, a farm, a preserve, a pet shop and so on, by use of the methods using a run 22, a groove 23, an aqueduct 24, an underground aqueduct 26 and so on, fresh water that receives a water supply from a water pipe 25 is drains tanks placed one after the other in order. The methods are the same as a drainage system such that liquid goes through a bank wall of a reservoir. [0004] With respect to any spontaneous overflow from a storage tank or a vessel without perforation of the tank or the vessel, the method provides an outflow that is done by a siphon action of a head 7 when a U-shaped tube in FIG. 1B or a hose is filled with water. When a storage tank or a container is large, the overflow is done after a siphon is filled with water by the use of a power pump at each start instead of by the use of manpower. [0005] As another usage of the siphon, it can be used as a measuring and draining apparatus, that is to say, the outlet pipe of the drain channel is formed as a siphon and the outlet of the outlet pipe is positioned at the same height as the horizontal surface of the liquid in the inside of the liquid storage tank and thereby the inside of the outlet pipe is always filled with liquid and residual volume in the inside of the outlet pipe is held at a given volume in order to drain liquid with accuracy and stability in volume from the fixed quantity of the cylinder of the exhaust pump. SUMMARY [0006] In an aqueduct 24 or an underground aqueduct 26 as shown in FIG. 5, a drainpipe connects with the tank wall of a variety of tanks or a farm/a preserve, a siphon such as a shape indicated in patent document 2 is installed over the tank wall because of the inferiority of stress variance strength and earthquake-resistance, there is a problem that siphon action is damaged and even if the water level of the inside of the tank goes up again spontaneous overflow is not effected when the water in the inside of the tank has run dry and water level is lower than the water level of an end of the siphon and air flows in the siphon. Therefore, a device with a power pump is used for starting the siphon. [0007] It is an object of the present invention to provide a siphon unit that reestablishes spontaneous overflow is effected rapidly and repeatedly by using siphon action without the use of a power pump every time the water level goes up in the inside of the tank. [0008] For the purpose of preventing breakdown of the siphon action when the perpetual overflow is stopped and air flows in the siphon, auxiliary pools in addition to a main pool at the position of the bottom of the auxiliary pools lower than the ends of the siphon and the position of the walls of the auxiliary pools higher than the ends of the siphon and lower than the wall of the storage tank is installed by manner of means indicated below and the inside of the siphon is always filled with liquid. [0009] A tube that upon expansion on a plane, assumes a W-shape is used and extends upward from the ends of a U-curved tube are used as auxiliary pools. Again the ends of the upward U-curved siphon tube are inserted and fixed to the upward openings of the auxiliary pools. [0010] Or, one of ends of the siphon tube is changed, and an auxiliary pool is put on both ends by the method of combining one end of the W-shape siphon tube previously described. In this way, when the liquid level of the storage tank is lower than the end of the siphon, flow in the siphon stops in the stage that the head of the liquid in the inside of the siphon and the liquid in the inside of both auxiliary pools are in balance and thereby the liquid in the inside of the siphon is perpetually prevented from flowing out. And when the liquid level of the storage tank goes up above the auxiliary pool by the pressure of the head, liquid flows spontaneously out of the storage tank through the siphon tube. [0011] To overflow to the position made a choice at random the auxiliary pool outside the storage tank must be connected with the outlet pipe. In the method of the W-shape, the extension is formed as a drainpipe by extending the top of the upward auxiliary pool downward and air is taken in from a vent bored at the top of the mountain-shaped connecting part to break off the relations of the water head between the auxiliary pool and the outlet pipe. [0012] Again in the method of the auxiliary pools a drainpipe is connected with the side wall of the auxiliary pool outside the storage tank at the height of the auxiliary pool in the storage tank and liquid is prevented from flowing out by arranging the wall of the auxiliary pool outside the storage tank higher than the connection portion and the upper portion is kept open. [0013] And the breakdown of siphon action caused by suctioning liquid in the auxiliary pool and the siphon tube out by suction power by the heads of the drain pipe is prevented. And to rapidly drain by making use of suck action caused by the head of the drain pipe, a valve for automatically shutting the vent or an opening to the atmosphere of the drain pipe only within the range of water level of liquid that exceeds the auxiliary pool where air is not inhaled from the suction port of the storage tank into the siphon tube by whirlpool of liquid is installed. [0014] It is possible to rapidly discharge liquid naturally and perpetually without the need of maintenance and power until the liquid in the siphon unit has evaporated and been lost and contribute to energy conservation. [0015] As for the tank the siphon unit is used, because it is not necessary to drill a hole in tank wall, the stress is not concentrated on the tank wall and because it is not necessary to connect each tank directly with the pipe, each tank can be placed independently. So then because each tank is strong against collapse, burst and leakage caused by the earthquake or the tremble, it is possible to set the chemical plant tank and the medicine tank to a smooth place, and to flow liquid out consecutively and to contribute for safety. [0016] By using the siphon, as shown in FIG. 4 compared with FIG. 5, the drainpipe can be easily set up without perforation of the processing existing and a cheap, free tank can be laid out. By adding the up and down device to the siphon unit it is possible to provide equipment having water level control, the flowing quantity control, and the measurement exhaust which can be done with ease and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS [0017] FIG. 1 is a schematic illustration of the invention. [0018] FIG. 2 is a schematic illustration of another embodiment of the invention. [0019] FIG. 3 is a schematic illustration showing the method of connecting the drainpipe. [0020] FIG. 4 is a schematic illustration where this siphon unit is arranged in the displayed tanks. [0021] FIG. 5 is a schematic illustration of a conventional drain method. [0022] FIG. 6 is an illustration of the water tank that uses this siphon unit. [0023] FIG. 7 illustrates a float valve for a vent because of the rapid drain. [0024] FIG. 8 is an illustration of the water surface type substitution valve that uses the vent extension tube for rapid drainage. DETAILED DESCRIPTION [0025] In addition to the main reservoir (the storage tank) auxiliary pools are installed to both ends of a siphon tube. And increased water that exceeds set water level can be perpetually drained out without the use of power pump and the need of maintenance as well as drain by perforation of the lower part of the storage tank. Example 1 [0026] The auxiliary pools are installed to both ends of the siphon tube that is used for drain in the following methods. [0027] One method is as follows. By the use of W-curved tube 1 that upon expansion on a plane assumes a W-shape in FIG. 1 risers of both ends of the tube are used as auxiliary pools at a position of the both ends that are lower than a mountain part of the center of the tube and the storage tank 5, higher than the trough part of the both sides. [0028] Another method is as follows. Both auxiliary pools 9 in FIG. 1A for always soaking the end of the siphon tube in water are fixed at the bottom position of the each auxiliary pool that is lower than a trailing end point of the siphon tube and at the wall position of the each auxiliary pool that is higher both ends of the siphon tube and lower than the wall of the storage tank. [0029] Another method is as follows. One end of a siphon tube is composed as shown in FIG. 3D and combined with the one side of the W-curved tube of the type of the above-mentioned W-curved tube in FIG. 3D. [0030] And then by balancing by head 2 and 10 of water of the inside the siphon tube and of the auxiliary pools, even if water level of the inside the storage tank goes down than water level 4 of the end part of the siphon tube inside the storage tank or than water level 12 as shown in FIG. 2, the flow finishing of water of the inside the siphon can be stopped, the breakdown of the siphon action can be prevented, and the siphon action can be maintained and managed perpetually. When water level of the inside the storage tank goes up and head 3 as shown in FIG. 1A or head 11 as shown in FIG. 2 is caused, water is made to flow out of the storage tank naturally through the siphon by pressure of the head. [0031] It is necessary to connect the drainpipe with the auxiliary pool to drain to an arbitrary position. By use of a method of installing a vent pipe 13 or a vent on the upper or “heaven” side of the junction of the drainpipe 14 as shown in FIG. 3C, or connecting a drainpipe 17 with the side of the auxiliary pool fixed to the siphon tube seen FIG. 3D and installing an opening 16, the water head relation between the auxiliary pool and the drainpipe can be severed, suction water head 15 of the drainpipe can be not applied to the inside of the siphon tube, water of the inside of the siphon tube and water of the inside of the auxiliary pool can be not inhaled fully into the drainpipe, thereby the siphon action can be maintained and managed, and overflow drain can be made at the proper water level of the inside the tank without the need of maintenance. [0032] In this siphon unit, according to circumstances, the glass tube where collecting of the air of the inside the siphon tube can be observed is used because air collects in the inside the siphon and doesn't function when the bubble of the handling liquid exceeds the limit and it doesn't set it up in the liquid that gasifies easily and the bubbling vicinity. [0033] Though curved tube in FIG. 1A is called a W-curved tube or W shaped siphon tube because upon expansion on a plane it assumes a W-shape. However for draining rain water to a water tank 32 at a given water level as indicated in FIG. 6 it is possible to arrange siphon tube 27 portion, an auxiliary pool 28 of a pipe rising in the tank, an auxiliary pool of a pipe rising outside the tank 29, a drainpipe 30, and a vent 31 in a free style within the range where the principle is not ruined to match it to a tank or surrounding circumstances. [0034] The following method by which the processing installation can be done at a low price and easily only by using the pipe is recommended. W-curved tube 1 in FIG. 1A that upon expansion on a plane assumes a W-shape compared with FIG. 2 is recommended. Moreover, to connect the drainpipe that can drain to an arbitrary position, the auxiliary pool method using only the pipe in FIG. 3C that is simpler than a device shown in FIG. 3D is recommended. The method is shown in FIG. 4. The water supplied by the water pipe is drained to the tanks one after the other where a W-curved tube 20, a vent pipe 18, and a drainpipe 19 were set up. When existing tanks are arranged and constructed, the method in FIG. 4 is far simpler compared with the method of perforation of the storage tank in FIG. 5. [0035] When the vent diameter of the junction of the auxiliary pool and the drainpipe is too small air to sever the suck action of the drainpipe that applies to the siphon tube cannot finish entering, the water of the inside the siphon is completely sucked out with the drain pipe and siphon action is broken and siphon action cannot be persistently maintained. That is, when head in FIG. 1 is enough and an effective diameter of the air path of vent pipe in FIG. 13 is small the suck action of the head 15 of the drainpipe 14 indicated in FIG. 3 works, and thereby there is an advantage in which the drain speed is quickened. However, it is necessary to expand an effective vent diameter of the air path to obtain an amount of necessary air before the water level of the tank falls, the head in FIG. 1 decreases, it approaches the water level 4, air with the whirlpool of water is inhaled into the unit, and thereby siphon action breaks. [0036] The electric motor operated valve with an electric water level sensor can be selected. But The float valve 34 in FIG. 7 is a vent valve for the rapid drain which is installed to make use of the suction and drainage capability of the head 15 of FIG. 3C according to the water level 35 of the tank. [0037] The state to shut the valve is indicated in FIG. 7E, and the state to open the valve is indicated in FIG. 7F. To open the valve from the adsorption of the vacuum pressure, spring 36 for valve opening/closing is installed. [0038] It is possible to detach the vent pipe from the float valve unit by the screw or by lightly press-fitting. And a valve 45 of the drainpipe that shuts when the supply of priming water is done from the air pipe to the whole unit is installed in the bottom part of the drainpipe at the first stage of the installation. [0039] The method of the water surface type vent opening and shutting function pipe that inserts a vent extension tube 38 of FIG. 8 in the tank can be applied in place of the valve and there is simplicity and a cost performance. [0040] When the surface of water inside the tank closes the vent extension tube and rapid drain starts by using a siphon that is united with the drainpipe as indicated in FIG. 1B in place of the action keeping type siphon action and the vent tube is shut at the water level 40 of FIG. 8 for a split second, as the inside of the drainpipe 39 is explained in FIG. 8, the water head of the drainpipe is not caused because water falls along inner wall with the air path from top to bottom left. Therefore, it is likely to fall into the state that cannot be high-speed drained even if rising up to the water level 41 in the tank like being a little flowing quantity. [0041] Consequently, like a slit 42 of FIG. 8H, by cutting the side of the end of the vent extension pipe, as the water level in the tank goes up, ventilation is shut gradually, and the pulsating flow is generated in the current of air of the vent tube. In a word, as the inside of the drainpipe is explained in FIG. 8H, the water level section 43 where pulsating flow of air bubble mixing is caused is temporarily necessary. [0042] The water level section of FIG. 8H that enables rapid drain by making the best use of the suction head of the drainpipe full is the level in the tank as shown in FIG. 3C. However, in the water level section 43, a gurgling sound of moving water is generated. Then when the sound is bothersome a float valve 34 of FIG. 7 is recommended. [0043] When the rapid drain function is installed like this it is necessary to consider the balance of an amount of the water supply in the tank, a head of the drainpipe, and a flowing quantity effective diameter of the entire unit because in both the float valve type for the vent and the alternative surface of the water type, as explained ahead, if the valve is shut momentarily the situation not rapidly drained happens, and if the drainpipe is too large the same thing occurs, and if the drainpipe is too small the drain capability reduces from flow resistance. [0044] In the tanks of FIGS. 7 and 8, a draining W-shape siphon 42 that uses a polyvinyl chloride (PVC) PVCφ16 pipe for water service, a vent pipe 43 that uses a PVCφ16 pipe, and a drainpipe that uses a PVCφ13 pipe of length of 1 m that corresponds to suction head of FIG. 3 are arranged and the drain capability was tested. As a result, it was confirmed that this siphon unit has 20 l/m drain capability. Also, it was confirmed that this siphon unit has performances as well as the method by perforation of the lower part of the storage tank for connecting with a drainpipe. [0045] Though the drain capability changes depending on the kind of the thickness of a flowing quantity effective diameter of the tube where the unit is composed and the thrown liquid, these can be used for the dam, the reservoir, the tank, the container, the beaker, and also for the liquids other than the water such as oil, the melt, the chemical, the medicine, the melting plastic, and the melting metals widely multipurpose. [0046] Because it installs on an easy taking the place of the engine pump and it is possible to detach it, this siphon unit is used for the irrigation water supply of millrace U character ditch and the rice field without holes. [0047] Because the amount of the supply from the first tanks to the second tank can be controlled by adding the going up and down device to either of this unit or the first tank, this siphon unit is used for the mixture tank of a liquid material or the reagent. [0048] For ease of reference, the following elements are numbered in the specification and drawings: 1 W-curved tube 2 head 3 head 4 water level 5 a storage tank 6 U-shaped tube 7 head 8 water level 9 an auxiliary pool 10 head 11 head 12 water level 13 vent pipe 14 drainpipe 15 head 16 opening 17 drainpipe 18 vent pipe 19 drainpipe 20 W-curved tube 21 water pipe 22 run 23 groove 24 aqueduct 25 water pipe 26 underground aqueduct 27 siphon tube 28 auxiliary pool (riser in the inside of the tank) 29 auxiliary pool (riser in the outside of the tank) 30 drainage 31 vent (hole) 32 water tank 33 water level of maintenance 34 float valve 35 water level 36 spring to open and close the valve 37 valve 38 vent extension tube 39 drainpipe 40 water level 41 water level 42 slit 43 water level section 44 water level section | Summary: An action keeping siphon unit includes a curved tube that upon expansion on a plane, assumes a W-shape is used as a siphon. Start-up portions on both sides thereof are used as auxiliary pools. Even when the water level in storage tank lowers to the bottom, a balance is kept by the heads of water held in the auxiliary pools to thereby stop any water flow break in the siphon, enabling perpetual prevention of breakdown of siphon action. Every time the water level in the storage tank once more goes up above the auxiliary pools and set water level, by the state of the water head similar to that of the siphon tube. | 4,732 | 146 | big_patent | en |
Summarize: A San Diego man convicted of identity theft and extortion after posting more than 10,000 sexually explicit photos of women to his so-called "revenge porn" website was sentenced on Friday to 18 years behind bars. NBC 7's Gene Cubbison reports. (Published Friday, April 3, 2015) A San Diego man convicted of identity theft and extortion after posting more than 10,000 sexually explicit photos of women to his so-called "revenge porn" website was sentenced on Friday to 18 years behind bars. The sentencing of Kevin Bollaert ended an all-day hearing where a number of victims told of the humiliation inflicted by his website. Bollaert burst into tears as he listened to testimony from his mother and victims. The sentence was at the high end of the range; Bollaert faced a maximum of 20 years. In explaining his punishment, the judge noted that he stacked the sentencing terms based on the multiple victims. Considering credits for good behavior, Bollaert could be eligible for parole after 10 years, the judge noted. Man Sentenced in Revenge Porn Case Kevin Bollaert was sentenced in San Diego on April 3, 2015, in a "revenge porn" case in which he posted thousands of nude and sexually explicit photos of women online without their permission. He's one of the first to be prosecuted under California's anti-revenge porn law. NBC 7's Liberty Zabala reports. (Published Friday, April 3, 2015) Bollaert also must pay $10,000 in restitution. It was the first case of its type in the United States, and California was the first state to prosecute someone for posting humiliating pictures online. Bollaert was convicted of 27 counts of identity theft and extortion in connection to the thousands of photos posted online. Once they were published, Bollaert would then demand hundreds of dollars from individuals to remove their photos through a second website he owned. Prosecutors called Bollaert "vindictive" and claimed he took pleasure out of hurting his female victims with the internet being his "tool of destruction." In court Friday, his parents told the judge their son has said he has shown remorse. "He has said many times he wishes he never made the website...If he could go back and change it all, he would," they said in a statement to the court. Revenge Porn Defendant Breaks Down in Court NBC 7's cameras were in court to capture the defendant as his parents spoke to a judge. This is video from the morning session. (Published Friday, April 3, 2015) One after another victims shared how they were damaged by Bollaert's actions. "It's just broken me on a level that's not describable," one woman told the court. "The only thing I have left is shame and anger." Another explained how she is haunted by her photos being made public, saying,"If someone looks at me? Are they remembering me?" She also described her experience as a daily struggle. A third victim said she has a hard time acknowledging Bollaert as a human being. The case centered on a now defunct website called YouGotPosted.com, created by Bollaert so ex-husbands and ex-boyfriends could submit embarrassing photos of victims for revenge. The photos also linked to victims’ social media accounts. Prosecutors say those who wanted to get the pictures taken down were redirected to another one of Bollaert's sites, ChangeMyReputation.com. There, the victims were charged $300 to $350 to have their photos removed. Revenge Porn Case Called a Landmark Kevin Bollaert, 27, has been charged with 31 felony counts for allegedly posting nude photos of women online, then charging the women to have the photos removed. NBC 7’s Megan Tevrizian talks with defense attorney Lindsey Mercer about why this is being called a landmark cases and what it could mean for similar cases going forward. (Published Sunday, Dec. 15, 2013) State law prohibits anyone from putting identifiable nude photos online after a breakup, punishable with $1,000 or six months in jail. Crawl of outlinks from wikipedia.org started March, 2016. These files are currently not publicly accessible. Properties of this collection. It has been several years since the last time we did this. For this collection, several things were done: 1. Turned off duplicate detection. This collection will be complete, as there is a good chance we will share the data, and sharing data with pointers to random other collections, is a complex problem. 2. For the first time, did all the different wikis. The original runs were just against the enwiki. This one, the seed list was built from all 865 collections. | Summary: The man behind a "revenge porn" website was sentenced yesterday to 18 years in prison for posting thousands of private photos and charging women to have them removed, the San Diego Union-Tribune reports. Kevin Bollaert, 28, was convicted last month of 27 felony counts-21 for identity theft and six for extortion-but still claimed during sentencing that he ran the now-defunct UGotPosted.com without knowing it was illegal. A prosecutor disagreed, saying "this man was told time and time again, 'You are ruining my life.'" Indeed, victims testified in a San Diego courtroom to the pain they had endured: "It's just broken me on a level that's not describable," said one of the women, NBC San Diego reports. "The only thing I have left is shame and anger." Bollaert often received the images from angry ex-boyfriends, then posted them and linked each image to a social-media page belonging to the victim. The women received all kinds of comments, from threats to come-ons, and could only have the images taken down by paying up to $350 at another Bollaert site, ChangeMyReputation.com. Bollaert admitted that he made $900 monthly from Web advertising and $30,000 by charging women to have images removed. "I don't know, dude. Like, it was just fun," read the judge in quoting Bollaert's words to an investigator. "At the beginning it was kind of fun and entertaining, but now it's kind of ruining my life." True enough: Bollaert will have serve at least half his sentence, pay a $10,000 fine, and $15,000 more to his victims, the LA Times reports. | 1,120 | 409 | multi_news | en |
Summarize: Hakimi già è andato via, Lukaku sta per seguirlo, ma la diaspora degli uomini simbolo dell'Inter scudettata potrebbe non essere finita qui, come in un incubo senza fine. Un incubo in cui i tifosi nerazzurri si sono ritrovati appena qualche settimana dopo aver festeggiato il trionfo della truppa di Antonio Conte. L'addio clamoroso del tecnico leccese assume nuova luce ogni giorno che passa: una decisione evidentemente presa non a cuor leggero e con tutti gli elementi necessari per valutarla bene. Conte conosceva già il finale della storia e non voleva mettere la sua firma su una sceneggiatura così orrenda. D'altronde se neanche la dichiarazione d'amore al 100% nerazzurro da parte di Romelu Lukaku – il gigante buono, il trascinatore, il leader carismatico – può essere una certezza, allora è davvero lecito aspettarsi di tutto: ovvero che l'Inter possa cedere anche Lautaro Martinez. Il punto è sempre lo stesso: decidere quale deve essere il livello di ‘indecenza' delle offerte per renderle irrinunciabili. Il Chelsea con Lukaku ha fatto all-in, arrivando a cifre mostruose, da record di tutti i tempi per l'Italia, mettendo anche sul tavolo del belga un ingaggio fuori mercato per la Serie A. | Summary: Sembra un incubo per la squadra che ha appena vinto lo Scudetto in Serie A, ma l'Inter non sembra in grado di resistere agli attacchi per i suoi gioielli. Sono assalti condotti con quantità di denaro imbarazzanti: perso Hakimi finito al PSG, in uscita anche l'uomo simbolo Lukaku diretto al Chelsea, adesso si rifà sotto l'Atletico Madrid per Lautaro Martinez. Anche in questo caso con un'offerta che può diventare 'irrinunciabile'. | 290 | 107 | fanpage | it |
Write a title and summarize: SECTION 1. MODIFICATIONS TO CARBON DIOXIDE SEQUESTRATION CREDIT. (a) Allocation and Certification of Credit.-- (1) In general.--Subsection (e) of section 45Q of the Internal Revenue Code of 1986 is amended to read as follows: ``(e) Limitation.-- ``(1) Allocation limitation.--No credit shall be allowed under subsection (a) with respect to qualified carbon dioxide captured by carbon capture equipment at a qualified facility for the amount of qualified carbon dioxide captured by such carbon capture equipment in excess of-- ``(A) the portion of the national limitation allocated with respect to such carbon capture equipment under subsection (f), over ``(B) the amount of qualified carbon dioxide captured by such carbon capture equipment during periods after July 31, 2013. ``(2) National limitation.--For purposes of paragraph (1)(A), the national limitation is the excess of-- ``(A) 75,000,000 metric tons of qualified carbon dioxide, over ``(B) the number of metric tons of qualified carbon dioxide captured before August 1, 2013, for which a credit under subsection (a) was allowed.''. (2) Allocation and certification.--Section 45Q of such Code is amended by adding at the end the following new subsection: ``(f) Allocation for and Certification of Carbon Capture Projects.-- ``(1) Establishment of procedures.--Not later than July 1, 2013, the Secretary shall establish, by regulation, processes and procedures-- ``(A) for allocating the national limitation under subsection (e)(2) to projects for placing carbon capture equipment in service at qualified facilities, and ``(B) for certifying projects for which an allocation has been made under subparagraph (A). ``(2) Allocations.-- ``(A) Application.--Each applicant for an allocation under this subsection shall submit an application to the Secretary under such terms and conditions as are established by the Secretary in regulations. ``(B) Priority.--The Secretary shall rank applications received under subparagraph (A) in the following order: ``(i) Applicants with applications received by the Secretary on an earlier date shall be given higher priority than applicants with applications received on a later date. For purposes of this clause, any application received before the date that is 30 days after the procedures and processes described in paragraph (1) are established shall be considered to have been received on such date. ``(ii) In the case of applications received on the same date, those applicants concurrently applying for certification shall be given higher priority. ``(iii) In the case of applications received on the same date and concurrently applying for certification, those projects with the earlier date by which construction commenced shall be given higher priority. ``(C) Allocation to applicants.--Subject to subparagraph (D), the Secretary shall allocate tonnage to each applicant-- ``(i) based on the amount requested on the application, and ``(ii) in order of the rank of the application under subparagraph (B), until the amount of tonnage available under this section is exhausted. Projects for which no or a partial allocation is made shall retain their ranking and shall be eligible to receive an allocation of tonnage previously allocated that is forfeited or recaptured. ``(D) Limitation.--The Secretary may not allocate to any project more than the lesser of-- ``(i) the number of metric tons of qualified carbon dioxide projected to be captured at the qualified facility under the project during the 10-year period beginning on the date on which such project is placed in service, ``(ii) the number of metric tons of qualified carbon dioxide projected to be captured at the qualified facility under the project-- ``(I) which are subject to a written, binding contract for disposal in secure geological storage (whether or not used as a tertiary injectant), or ``(II) for which there is a plan for such disposal by the applicant, or ``(iii) 15,000,000 metric tons of qualified carbon dioxide. ``(E) Reduction for prior credits.--The amount of any allocation under subparagraph (C) to any project shall be reduced by the number of metric tons of carbon dioxide captured by the carbon capture equipment with respect to such project before August 1, 2013, for which a credit was allowed under subsection (a). ``(3) Certification.-- ``(A) In general.--No credit shall be allowed under subsection (a) with respect to any project for using carbon capture equipment to capture qualified carbon dioxide at a qualified facility before the date on which such project is certified under this paragraph. ``(B) Application for certification.--Each project which is allocated a portion of the national limitation shall submit an application for certification to the Secretary containing such information as the Secretary may require. Such application shall be submitted-- ``(i) not later than-- ``(I) 6 months after the date on which such project receives an allocation, and ``(II) 30 days after the later of the date on which the regulations, processes, and procedures are established under paragraph (1) or the construction start date, and ``(ii) not earlier than the construction start date. For purposes of this subparagraph, the term `construction start date' means the earlier of the first date on which physical work on the project of a significant nature is undertaken or the date by which 5 percent or more of the total cost of the project has been spent. ``(C) Revocation of certification.-- ``(i) Materially inaccurate representations.--The Secretary may revoke a certification under this paragraph if the Secretary determines that an applicant has made a materially inaccurate representation with respect to the project. ``(ii) Failure to timely place equipment in service.--A certification under this paragraph shall be revoked in any case in which carbon capture equipment with respect to the project is not placed in service-- ``(I) before the date which is 5 years after the date on which the allocation was issued, in the case of a new industrial facility, or ``(II) before the date which is 3 years after the date on which the allocation was issued, in the case of a modification of an existing industrial facility. ``(D) Reallocation.--In any case-- ``(i) in which a certification is revoked under subparagraph (C), or ``(ii) in which a taxpayer to whom an allocation is made under paragraph (2) fails to obtain certification for a project under this paragraph, the amount of national limitation which was allocated to such project under paragraph (2) shall be reallocated under such rules as established by the Secretary under regulations. ``(4) Public disclosure.-- ``(A) In general.--The Secretary shall, within 30 days of making any allocation, certification, revocation, or change in the ranking of projects, publicly disclose the amount of such allocation, a description of the project for which such allocation, certification, or revocation was made, and the change in the ranking of projects, as the case may be. ``(B) Annual report.--The Secretary shall issue an annual report summarizing credits allocated and available for allocation.''. (3) Conforming amendments.-- (A) Paragraph (2) of section 45Q(c) of such Code is amended by inserting ``which is part of a project which is certified under subsection (f)(3)'' after ``carbon capture equipment''. (B) Paragraph (3) of section 45Q(c) of such Code is amended by striking ``which'' and inserting ``at which such carbon capture equipment''. (b) 10-Year Credit Limitation.--Section 45Q(a) of the Internal Revenue Code of 1986 is amended-- (1) in paragraph (1)(A), by inserting ``during the 10-year period beginning on the later of the date on which the carbon capture equipment described in subsection (c)(1) is placed in service or the date on which the project with respect to such carbon capture equipment was certified under subsection (f)(3)'' after ``qualified facility'', and (2) in paragraph (2)(A), by inserting ``during the 10-year period beginning on the later of the date on which the carbon capture equipment described in subsection (c)(1) is placed in service or the date on which the project with respect to such carbon capture equipment was certified under subsection (f)(3)'' after ``qualified facility''. (c) Definition of Carbon Capture Equipment.--Section 45Q(d) of the Internal Revenue Code of 1986 is amended by adding at the end the following new paragraph: ``(8) Carbon capture equipment.--The term `carbon capture equipment' means equipment to capture and pressurize qualified carbon dioxide.''. (d) Credit Allowed to Taxpayer Performing Carbon Capture.-- (1) In general.--Paragraph (5) of section 45Q(d) of the Internal Revenue Code of 1986 is amended to read as follows: ``(5) Person to whom credit is allowable.-- ``(A) In general.--Except as provided in subparagraph (B) or in regulations prescribed by the Secretary, any credit under this section shall be allowed to the taxpayer who-- ``(i) captures the qualified carbon dioxide, and ``(ii) through contract or otherwise, disposes of the qualified carbon dioxide in a manner meeting the requirements of paragraph (1)(B) or (2)(C) of subsection (a), as the case may be. ``(B) Election to allow credit to person disposing carbon dioxide.--If the person described in subparagraph (A) makes an election under this subparagraph in such manner as the Secretary may prescribe by regulations, the credit under this section-- ``(i) shall be allowable to the person that disposes of qualified carbon dioxide in a manner meeting the requirements of paragraph (1)(B) or (2)(C) of subsection (a), as the case may be, and ``(ii) shall not be allowable to the person described in subparagraph (A).''. (2) Conforming amendments.-- (A) Section 45Q(a) of such Code is amended by striking ``by the taxpayer'' each place it appears in paragraph (1)(B), (2)(B), and (2)(C). (B) Section 45Q(c) of such Code, as amended by subsection (a), is amended by striking paragraph (1) and redesignating paragraphs (2) and (3) as paragraphs (1) and (2), respectively. (e) Rules Relating to Credit Recapture.--Paragraph (6) of section 45Q(d) of the Internal Revenue Code of 1986 is amended by adding at the end the following new sentence: ``Notwithstanding section 7805(b), any regulation issued pursuant to this paragraph shall apply only with respect to qualified carbon dioxide captured or disposed of after the date on which such regulation is filed with the Federal Register.''. (f) Effective Date.--The amendments made by this section shall apply to carbon dioxide captured after July 31, 2013. | Title: A bill to amend the Internal Revenue Code of 1986 to modify the credit for carbon dioxide sequestration Summary: Amends the Internal Revenue Code, with respect to the tax credit for carbon dioxide sequestration, to: (1) establish a national limitation for such credit based upon metric tons of qualified carbon dioxide (defined as carbon dioxide captured from an industrial source that would otherwise be released into the atmosphere as industrial emission of greenhouse gas and that is measured at the source of capture and verified at the point of disposal or injection); (2) direct the Secretary of the Treasury to establish processes and procedures for allocating the national limitation and for certifying projects for which an allocation has been made; (3) impose a 10-year limitation period for such credit; (4) identify the primary taxpayer eligible to claim such credit as the taxpayer who captures the qualified carbon dioxide and disposes, through contract or otherwise, of the qualified carbon dioxide in a specified manner; and (5) provide for the transferability of such credit. | 2,666 | 226 | billsum | en |
Summarize: Rationale for Program The origin of the Trade Adjustment Assistance for Farmers (TAAF) program can be traced back to a 2000 Department of Labor report recommending that a separate program be enacted "to assist agricultural producers and workers affected adversely by imports" if the objective is to assist them to remain in their current occupations. The report described the existing trade adjustment assistance (TAA) programs that provided (1) limited technical assistance to help business firms (including some that produced agricultural and food products) regain economic competitiveness or to shift into producing other goods, and (2) training assistance to workers (including those employed by some agricultural firms) to facilitate their transition into other occupations. It noted that the provision of direct financial assistance (such as income supplements) to farmers, or efforts to financially enable them to continue producing the commodity adversely affected by imports rather than help them adjust to employment in other sectors, would be inconsistent with the objectives of the then-existing TAA programs. Observers stated that farmers and ranchers typically did not qualify for the TAA workers program because they were self-employed (and thus rarely were eligible for unemployment benefits) and were less likely to want to be retrained for a new occupation (particularly if earning income from producing other crops or from non-farm sources). Others pointed out that agricultural producers who are most likely to be affected by import surges are those producing a commodity that receives little or no price protection and does not receive direct payments under traditional farm subsidy programs. Frequently cited at the time was the impact of increased competition that U.S. fruit and vegetable growers, as well as livestock producers, have encountered due to imports from Mexico and Canada under the North American Free Trade Agreement. Overview of TAAF Program The Trade Act of 2002 established a new Trade Adjustment Assistance for Farmers program by amending the Trade Act of 1974 ( P.L. 93-618 ). The U.S. Department of Agriculture's (USDA's) Foreign Agricultural Service (FAS) is the lead administrative agency for the TAAF program, with responsibility for certifying eligible commodities and producer groups. USDA's Farm Service Agency (FSA) has responsibility for processing and approving individual applications for assistance under TAAF, and for disbursing cash payments to eligible producers. A third USDA agency, the National Institute for Food and Agriculture (NIFA), provides training and technical assistance to producers who are approved for TAAF benefits. As amended by the enacted 2009 economic stimulus package ( P.L. 111-5, Division B, Subtitle I), the program assists agricultural producers who have been adversely affected by competition from imports of a commodity that they produce. An "agricultural commodity producer" is defined as a "person that shares in the risk of producing an agricultural commodity and that is entitled to a share of the commodity for marketing, including an operator, a sharecropper, or a person that owns or rents the land on which the commodity is produced," or a person who reports a gain or loss on a federal income tax return from "the trade or business of fishing." Support is available in the form of enhanced technical assistance and seed money to enable a producer to formulate and implement a business adjustment plan. Producers of raw and natural agricultural commodities (crops, livestock, farm-raised aquatic products, and wild-caught seafood that competes with aquaculture products) and of "any class of goods within an agricultural commodity" must follow a two-part process to receive benefits. First, a producer group must be certified by USDA as eligible to apply for program benefits (see " Requirements for a Commodity Group to Be Certified "). Second, if the group is certified, individual producers in that group must meet certain requirements to be approved to receive technical assistance and cash payments (see " Individual Producer Eligibility Requirements " and " TAAF Program Benefits "). Requirements for a Commodity Group to Be Certified A group of agricultural producers can petition the Secretary of Agriculture to be certified as eligible to participate in the TAAF program (i.e., to qualify for benefits). To certify a commodity group, the Secretary must determine that the increase in imports of the agricultural commodity produced by members of the group "contributed importantly" to at least a 15% decline in the national average price, quantity of production, or value of production or cash receipts of the commodity. In making a determination, the Secretary must compare the volume of imports of "articles like or directly competitive with the agricultural commodity" produced by the group in the marketing year in which the petition is filed, to the average volume of imports in the three preceding marketing years. The addition of two other qualifying factors—"quantity of production" and "value of production/cash receipts"—besides price gives the Secretary greater flexibility in determining if a commodity group is eligible to access program benefits. The Secretary then has 40 days to make a determination on a group's petition. Individual Producer Eligibility Requirements If the Secretary certifies that a group qualifies for assistance, each producer in the group has 90 days to apply for TAAF benefits. To be eligible, an individual producer must show in the application submitted to USDA that (1) the agricultural commodity was produced in the year covered by the group's petition, and in at least one of the three preceding marketing years; (2) the quantity of the commodity produced in that year has decreased compared to the amount produced in a previous year, or the price received for the commodity in that year has decreased compared to the average price received in the preceding three marketing years; and (3) no cash benefits were received under the TAA for Workers and TAA for Firms programs, nor were benefits received based on producing another commodity eligible for TAAF assistance. The reauthorization of TAAF through FY2021 under P.L. 114-27 does not alter the eligibility requirements for commodity groups or individual producers that existed heretofore. TAAF Program Benefits The changes enacted in 2009 refocus the TAAF program by (1) making technical assistance available to an eligible producer, and (2) providing financial resources so that a producer can put into effect a business plan to make adjustments in the operation. A producer approved for the TAAF program is entitled to receive initial technical assistance (TA) to improve competitiveness in the production and marketing of the commodity certified to receive benefits. Such assistance is to include information on what steps could be taken to improve the yield and marketing of that commodity, and on exploring the feasibility and desirability of substituting one or more alternative commodities for the one being produced. USDA can provide supplemental assistance to cover reasonable transportation and subsistence expenses that a producer incurs in accessing initial technical assistance if provided in a location outside a normal commuting distance. A producer who completes this initial phase is eligible to participate in intensive technical assistance. This includes training courses to assist the producer in improving the competitiveness of the same commodity or an alternative commodity, and financial assistance to develop an initial business plan based on the courses completed. USDA is required to approve a producer's initial business plan if it reflects the skills gained by the producer through the courses taken. Further, this plan must demonstrate how the producer will apply these skills to his circumstances. If the plan is approved, the producer is entitled to not more than $4,000 to implement this plan, or to develop a long-term business adjustment plan. A producer who completes the intensive phase and whose initial business plan has been approved is then eligible for assistance to develop a long-term business adjustment plan. USDA is required to approve this adjustment plan if it includes steps calculated to materially contribute to the producer's economic adjustment to changing market conditions, takes into account the interests of the workers employed by the producer, and demonstrates that the producer will have sufficient resources to implement the business plan. If approved, the producer is entitled to $8,000 to implement this long-term plan. Limitations on Producer Financial Assistance The amount of assistance that a producer can receive to implement both the initial business plan and the long-term business adjustment plan is limited to $12,000 in the 36-month period after USDA has certified producers of the commodity as eligible for TAAF benefits. Further, TAAF-eligible producers cannot receive cash benefits under any other TAA program. An applicant is ineligible for TAAF assistance in any year in which his average adjusted gross income exceeds the level specified in Section 1001D of the Food Security Act of 1985 as amended (i.e., $500,000 of non-farm income, or $750,000 of farm income, depending on the details of the applicant's involvement in a farm operation, beginning with the 2009 crop year). Written Notices to Producers The Secretary of Agriculture is required to provide written notice to each agricultural commodity producer in a group certified as eligible to receive benefits. A notice stating the benefits available to certified producers must also be published in newspapers of general circulation in the areas in which such producers reside. Program Coordination When notified by the International Trade Commission (ITC) that it has begun a safeguard investigation of a particular agricultural commodity, the Secretary of Agriculture is required to conduct a study of (1) the number of agricultural commodity producers who are producing a competitive commodity who have been or are likely to be certified eligible for TAAF, and (2) the extent to which existing programs could facilitate producers' adjustment to import competition. A safeguard (e.g., in the form of additional tariffs, expanded quota, or another restriction on imports) is intended to provide relief from the adverse impact of imports when temporary protection will enable the domestic sector (i.e., producers) to make adjustments to meet import competition. Within 15 days after the ITC has determined whether or not injury has occurred and reported its recommendations to the President, the Secretary must submit a report to the President on the USDA study's findings. History of TAAF Funding The Trade Act of 2002 ( P.L. 107-210 ) that established the TAAF program authorized and appropriated $90 million annually for FY2003 through FY2007 to operate the program. Under Section 1(c) of P.L. 110-89, Congress provided an appropriation of $9 million for TAAF for the first quarter of FY2008 (October 1 to December 31). Funding then lapsed until October 1, 2008, when Section 1887 of the American Recovery and Reinvestment Act of 2009 ( P.L. 111-5 ) authorized and appropriated $90 million in each of FY2009 and FY2010, and $22.5 million for the first quarter of FY2011 (October to December 2010). This provision also specified that funding shall cover the costs of administering the TAAF program. Congress temporarily extended funding for TAAF by providing an appropriation of $10.4 million for the period January 1, 2011, through February 12, 2011, in Section 101 of the Omnibus Trade Act of 2010 ( P.L. 111-344 ), but USDA viewed this six-week period as too short to implement another FY2011 program, so no activity occurred. Under Section 223 of the Trade Adjustment Assistance Extension Act of 2011 ( P.L. 112-40 ), Congress authorized $90 million in each of FY2012 and FY2013, and $22.5 million for the first quarter of FY2014 (i.e., October through December 2013). This provision, unlike those in the 2002 and 2009 authorizations, did not appropriate any funds. Because Congress did not subsequently appropriate funds, USDA did not announce TAAF programs for FY2012, FY2013, and the first quarter of FY2014. Authority to operate TAAF expired on December 31, 2013. Most recently, Section 403 of Title IV of the Trade Preferences Extension Act of 2015 ( P.L. 114-27 ) authorized TAAF to be appropriated $90 million each year for FY2015 through FY2021. As such, any program activity under TAAF will depend on the level of funding that Congress may appropriate. To date, no new funds have been appropriated. TAAF Program Implementation Because Congress in 2009 significantly revised TAAF's statutory provisions from those initially enacted, the text that follows describes how this program operated in the period before, and then in the period after, these changes. The break between periods reflects the lack of program authority in the January to September 2008 period. FY2003-December 2007 Activity under the TAAF in the FY2003-December 2007 period was much lower than authorized funding levels because of low producer participation and low payments, according to the Government Accountability Office (GAO). Of the $459 million authorized for the 5¼-year period through December 31, 2007, budget outlays totaled almost $49 million, according to USDA's Office of Inspector General (OIG) and USDA's Foreign Agricultural Service. This included $27.7 million in cash benefits paid to producers, $9.5 million for technical assistance, and $10.5 million for administrative costs ( Table 1 ). Of the 72 petitions filed by producer groups for assistance during the 5-year period that USDA received petitions, USDA certified or approved 30 groups ( Table 2 ). Shrimp and salmon producers accounted for most of the cash benefits paid out. Producers of Concord grapes, lychees, olives, wild blueberries, fresh potatoes, Florida avocadoes, snapdragons, and catfish were among other producer groups that USDA certified to be eligible for assistance ( Table 3 ). About 8,400 producers qualified for cash payments ( Table 2 ). FY2009 to Present Administrative Actions On August 25, 2009, USDA's Foreign Agricultural Service published a proposed rule to establish procedures for a group to request certification of eligibility, and for individual producers to apply for technical assistance and cash benefits, under the amended TAAF program. On March 1, 2010, USDA issued the TAAF interim rule and announced that it would immediately begin to implement the FY2010 program. This allowed producer groups to submit petitions to be certified for eligibility, which, if approved, permit individual members of a group to apply for program benefits. For FY2010, USDA accepted petitions through April 14, 2010. It certified 3 of the 11 petitions submitted by producer groups ( Table 2 ). If a petition was approved, eligible producers had to file applications for assistance within 90 days of the certification. On May 21, 2010, USDA announced that it would accept petitions for the FY2011 TAAF program through July 16, 2010. USDA in late September 2010 certified 7 of the 19 producer groups that submitted petitions ( Table 2 ). Eligible producers had until late December 2010 to file applications for assistance. Certifications and Producer Approvals With the 2009 changes to the TAAF program that eased the criteria for a producer group to be certified and for individual producers to be approved for program assistance, more of the provided funding has been used than in the FY2003-December 2007 period. USDA committed $127 million of the almost $203 million authorized for the 2¼-year period ending December 2010. This included $81.1 million in cash benefits and training costs for producers, $34.0 million for developing the technical assistance resources to be used to provide training, and $12.0 million for administrative costs ( Table 1 ). Funds obligated under the 2009 amendments represented 63% of authorized funding. (For comparison, outlays in the earlier period of FY2003 through December 2007 accounted for 10% of funding authority.) Of the 30 petitions filed since FY2009 by producer groups seeking certification (i.e., eligibility to qualify for assistance), USDA certified 10 groups. These included producers of shrimp, catfish, lobsters, asparagus, and wild blueberries ( Table 3 ). USDA subsequently approved about 4,500 producers for training assistance and cash benefits in FY2010. Another 5,700 applications were approved under the FY2011 program ( Table 2 ). USDA data show that most of the benefits under both years' programs flowed to shrimp producers in Alaska and along the Gulf and southern Atlantic states. As of late FY2012, of the 10,242 producers approved in FY2010 and FY2011 to receive program benefits, 80% had completed the intensive 12-hour training phase, 79% had completed their initial business plan, and 61% had completed their long-term business plan. Benefits to individual producers are based on the amount of funds authorized each year and are available only to those approved to receive technical and financial assistance. For the FY2010 program, approved producers were eligible for $12,000 in cash payments (see " TAAF Program Benefits," above, for details). But because only $22.5 million were available in the shortened FY2011 period for a larger number of approved applicants than in the previous year, each producer received pro-rated cash payments. During FY2012, FY2013, and FY2014, USDA continued to disburse financial assistance to producers approved to receive benefits under the FY2010 and FY2011 programs as they subsequently met certain benchmarks. GAO Report As required by Section 1894 of P.L. 111-5, the Government Accountability Office (GAO) in mid-July 2012 reported on the operation and effectiveness of the 2009 amendments made to the TAAF statute. It found that USDA certified relatively few commodities (five) under the changes made to the program, but that TAAF benefited most of the farmers and fishermen (over 10,200) who produced these certified commodities and had been approved to receive assistance. GAO discovered that the 2009 changes in the criteria used to determine whether a commodity can be certified were a factor leading to four of the five commodity certifications. For example, FAS under the pre-2009 criteria would not likely have been able to certify asparagus or shrimp solely on the basis of a decline in price. But in applying one new criterion—a decrease in the quantity produced of a commodity—as producers adjusted to increased imports, these two commodities were qualified to be certified. In reviewing how USDA implemented the program, GAO offered three recommendations: Require spouses of producers (i.e., those who share in the risk of producing an agricultural commodity) who may be eligible to apply for assistance to provide documentation on how they contribute to producing a certified commodity. This would address instances where USDA may have approved the applications of spouses who did not engage in producing a certified commodity, and thus had no assurance that TAAF assistance was appropriately targeted to intended recipients. Take steps to help ensure that any financial assistance payments made to producers are used for intended purposes (e.g., by requiring them to detail in their business plans how they plan to use these funds). This would address the acknowledgement made by USDA officials that some producers likely use the payments for unrelated expenses. Broaden the program's evaluation approach to help ensure that USDA can comprehensively evaluate the impact of the TAAF program on producers' competitiveness. GAO noted that the performance measures and surveys used by USDA do not measure quantifiable outcomes or cover all key areas of the program. To illustrate, conducting a final survey 6-12 months after producers complete the program does not allow for gathering insights into their perceptions of TAAF's long-term effectiveness. Also, USDA has not corroborated the results of surveys to help isolate the program's impact from other influences. USDA commented that it generally agrees with these recommendations, and that if a future TAAF program retains the same statutory requirements, it will consider specific ways to address them. USDA OIG Audit Called for Improved Oversight In an audit report of TAAF dated October 2013, the USDA's Office of Inspector General (OIG) identified a number of shortcomings in the administration of the TAAF program. The objective of the audit was to evaluate the internal controls established by FAS, FSA, and NIFA for administering the TAAF program, and to assess the program's policies and procedures. More specifically, the audit sought to determine whether (1) TAAF program recipients were eligible for program participation; (2) funds were properly obligated, distributed in a timely fashion, and accurately calculated; (3) program reporting requirements were met; and (4) oversight was sufficient to ensure that TAAF was administered in an accountable and equitable manner. A sampling methodology was developed to carry out this task. In brief, the OIG found the agencies did not have the appropriate controls in place to ensure that TAAF program participants were eligible, that payments were accurate, or that oversight was sufficient. The audit made a number of recommendations that follow from four key findings below. Agency responses to the findings and recommendations advanced by OIG are included in the audit report. 1. At the end of FY2009, FAS did not return unobligated TAAF program funds to the Department of the Treasury, nor did it provide evidence to show that all remaining funds were needed to meet future financial obligations. 2. FAS did not sufficiently analyze the documentation submitted by producer groups in support of their request for price pre-certification approvals for their commodities under a streamlining procedure. As a consequence, FAS applied the pre-price certification approvals in an overly broad manner, with the result that two of five such approvals that OIG examined did not meet the criteria established for approval. 3. Although FAS was the lead agency with oversight responsibility for the TAAF program, the agency failed to effectively monitor, or conduct reviews of, the two other agencies' day-to-day administration of the program, with the result that OIG identified 85 ineligible producers who participated in the TAAF program and who received approximately $284,000 in program benefits to which they were not entitled. 4. NIFA did not ensure that the TAAF program database was in compliance with federal information system security requirements for certification and accreditation. Major Trade Agreements Could Bring Adjustments Following the enactment of P.L. 114-26 —the law that provides the President with Trade Promotion Authority (TPA)—the Obama Administration concluded negotiations on the Trans-Pacific Partnership (TPP) regional free-trade agreement, which includes the United States and 11 other Pacific-facing nations, but to date the agreement has not been ratified by Congress or by other TPP countries. Meanwhile, negotiations between the United States and the European Union to conclude a Transatlantic Trade and Investment Partnership (T-TIP) agreement are ongoing. Considering the breadth of these two potential regional trade agreements, the economic diversity of the nations involved, and the broad range of U.S. agricultural and fishery products that might potentially be affected if TPP were to be implemented or if T-TIP were to be successfully concluded and implemented—or both—it is conceivable that some U.S. agricultural producers and fishermen might qualify for trade adjustment assistance under TAAF in the years ahead. Whether TAAF will be provided with funding to allow it to resume operations, and at what level, is for Congress to determine. In the event that Congress decides to appropriate funds to reactivate TAAF, Congress could provide oversight of the program in light of recommendations advanced by GAO and the USDA's Office of Inspector General. | Summary: The Trade Adjustment Assistance for Farmers (TAAF) program provides technical assistance and cash benefits to producers of farm commodities and fishermen who experience adverse economic effects from increased imports. Congress first authorized this program in 2002, and made significant changes to it in the 2009 economic stimulus package (P.L. 111-5). The 2009 revisions were aimed at making it easier for farmers and fishermen to qualify for program benefits, and provided over $200 million in funding through December 2010. Subsequently, P.L. 112-40 (enacted in October 2011) authorized $202.5 million through December 2013, but no new program activity has occurred since December 2010 for lack of appropriated funds. In June 2015, Congress passed H.R. 1295, the Trade Preferences Extension Act of 2015, authorizing TAAF through FY2021, and the President signed the bill into law on June 29, 2015, as P.L. 114-27. Any program activity would still be contingent on the appropriation of funds. The U.S. Department of Agriculture (USDA) is required to follow a two-step process in administering TAAF. First, a group of producers must be certified eligible to apply. Second, a producer in a certified group must meet specified requirements to be approved for benefits. To be certified, a group must show that imports were a significant cause for at least a 15% decline in one of three factors: the price of the commodity, the quantity of the commodity produced, or the production value of the commodity. Once a producer group is certified, an individual producer within that group must meet three requirements to be approved for program benefits. These include technical assistance with a training component, and financial assistance. A producer must show that (1) the commodity was produced in the current year and also in one of the previous three years; (2) the quantity of the commodity produced decreased compared to that in a previous year, or the price received for the commodity decreased compared to a preceding three-year average price; and (3) no benefits were received under any other trade adjustment assistance program. The training component is intended to help the producer become more competitive in producing the same or another commodity. Financial assistance is to be used to develop and implement a business adjustment plan designed to address the impact of import competition. From 2009 to 2011, USDA certified 10 of 30 petitions filed by producers of 5 commodity groups-shrimp, catfish, asparagus, lobster, and wild blueberries. USDA approved TAAF benefits for about 4,500 individual producers in FY2010, and for about 5,700 producers in FY2011. In a 2012 audit of TAAF, the Government Accountability Office recommended that USDA require spouses who apply for assistance to submit documentation on how they contribute to producing a commodity, take steps to ensure that the program's financial assistance component is used for intended purposes, and adopt a longer-term approach to evaluate its effectiveness. A 2013 audit by USDA's Office of Inspector General (OIG) identified several shortcomings in administering the program, including determining eligibility and providing effective oversight. The 2015 reauthorization of TAAF programs follows directly in the wake of the enactment of Trade Promotion Authority (TPA) legislation (P.L. 114-26) that President Obama had requested of Congress to facilitate the conclusion of regional free trade agreements, including the Trans-Pacific Partnership (TPP) with 11 other Pacific-facing nations. Under P.L. 114-27, TAAF is authorized to receive $90 million annually for FY2015 through FY2021, subject to annual appropriations. No new funding has been appropriated since the program's reauthorization. | 5,481 | 907 | gov_report | en |
Summarize: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a beating blade of a beater for use in cooking and, more particularly, to a novel construction of a beating blade of a beater capable of sufficiently stirring and efficiently beating materials such as an egg into froth in a short period of time. 2. Description of the Prior Art Various shapes of beating blades have been proposed and used up to now. For instance, in order to improve the beating or whipping efficiency, there has been used a comb-like blade having a plurality of slit-like openings in its surface. It is aimed that the chance of contact of the cooking material such as an egg with the blade is increased to improve the efficiency of trapping of air and hence the efficiency of beating or whipping. The beater incorporating this type of blades, however, cannot always ensure a good stirring efficiency because the cooking material tends to flow or move along the slit-like opening. SUMMARY OF THE INVENTION It is therefore a first object of the invention to provide a beating blade capable of efficiently beating or whipping the cooking material into froth. It is a second object of the invention to provide a highly efficient beating or whipping by making the cooking material interfere with the beating surface, through guiding the cooking material residing near the inner peripheral surface of a beater vessel to the central portion of the latter. It is a third object of the invention to provide a beating blade capable of generating a vertical flow of the cooking material in the vessel to render the beating or whipping more efficient. It is a fourth object of the invention to provide a beating blade with which the cooking material is prevented from passing through the area above the blade without dropping onto the latter, thereby to enhance the chance of the interference of the cooking material with the stirring surface. BRIEF DESCRIPTION OF THE DRAWINGS Various other objects, features and attendant advantages of the present invention will be more fully appreciated as the same becomes better understood from the following detailed description when considered in connection with the accompanying drawings in which like reference characters designate like or corresponding parts through the several views and wherein: FIG. 1 is a vertical sectional front elevational view of a beater having blades embodying the present invention; FIG. 2 is a perspective view of a beating blade incorporated in the beater as shown in FIG. 1; FIG. 3 is a cross-sectional view of the beating blade as shown in FIG. 2 by III; FIG. 4 is an enlarged sectional view of a portion of the beating blade as shown in FIG. 2; FIG. 5 is an enlarged perspective view of a mesh; FIG. 6 is a perspective view of a beating blade which is a second embodiment of the invention; FIG. 7 is a perspective view of a beating blade which is a third embodiment of the invention; FIG. 8 is a perspective view of a beating blade which is a fourth embodiment of the invention; FIG. 9 is a perspective view of a part of the beating blade as shown in FIG. 8; FIG. 10 is a sectional view of the beating blade as shown in FIG. 9 by X; FIG. 11 is a front elevational view of a beating blade which is a fifth embodiment of the invention; FIG. 12 is a perspective view of a beating blade which is a sixth embodiment of the invention; FIG. 13 is a perspective view of a beating blade which is a seventh embodiment of the invention; FIG. 14 is a perspective view of a beating blade which is an eighth embodiment of the invention; FIG. 15 is a perspective view of a portion of the beating blade as shown in FIG. 14; FIG. 16 is a sectional view of the portion of the beating blade as shown in FIG. 15 by XVI; FIG. 17 is a front elevational view of a beating blade which is a ninth embodiment of the invention; FIG. 18 is a perspective view of a beating blade which is a tenth embodiment of the invention; FIG. 19 is a longitudinal sectional view of the beating blade as shown in FIG. 18 by XIX; FIG. 20 is an enlarged longitudinal sectional side elevational view of a portion of the beating blade as shown in FIG. 18; FIG. 21 is an enlarged perspective view of a network and comb-teeth of the beating blade as shown in FIG. 18; FIG. 22 is a perspective view of a beating blade which is an eleventh embodiment of the invention; FIG. 23 is a longitudinal sectional view of the beating blade as shown in FIG. 2 by XXIII; FIG. 24 is a front elevational view of a beating blade in which a multiplicity of comb-teeth-like ribs are formed in the peripheral portion of the beating blade; FIG. 25 is a perspective view of a beating blade which is a twelfth embodiment of the invention; FIG. 26 is an enlarged perspective view of a portion of the beating blade as shown in FIG. 25; FIG. 27 is a perspective view of a beating blade which is a thirteenth embodiment of the invention; FIG. 28 is a side elevational view of the beating blade as shown in FIG. 27; FIG. 29 is a bottom plan view of the beating blade as shown in FIG. 27 with a portion thereof being removed; FIG. 30 is a front elevational view of a beating blade which is a fourteenth embodiment of the invention; FIG. 31 is a perspective view of the beating blade as shown in FIG. 30; FIG. 32 is a horizontal sectional view of a portion of the beating blade as shown in FIG. 30 by XXXII; FIG. 33 is a plan view of a portion of the beating blade as shown in FIG. 30; FIG. 34 is a bottom plan view of a portion of the beating blade as shown in FIG. 30; and FIGS. 35 and 36 are vertical sectional front elevational views of a beater illustrating the stirring action. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS A first embodiment of the invention will be described hereinafter with reference to FIGS. 1 to 5. Referring to these Figures, a reference numeral 1 denotes a casing of a beater. A motor 2 is fixed by means of screws 3 to one side of the casing 1 and is housed by the latter. A gear box 4 is fixed by means of screws 5 to the bottom of the casing 1 and is housed by the latter. The gear box 4 contains a transmission mechanism 6 including a plurality of gears operatively connected to the motor 2. An output shaft 8 connected to a final gear 7 of the transmission mechanism 6 projects out of the gear box 4 upwardly from the central portion of a vessel mounting portion 9 defined at the other side of the casing 1. The projecting end of the output shaft 8 has a coupling 10. A coupling 11 adapted to be releasably coupled to the coupling 10 is attached to the lower end of a blade shaft 12. The blade shaft is received in a watertight manner by a bore formed in the bottom of a vessel 13 which is detachably held in the vessel mounting portion 9. A beating blade member 14 is attached to the blade shaft 12. The blade member 14 is allowed to move in the axial direction of the blade shaft 12 along the latter, but is prevented from rotating relatively to the blade shaft 12. The beating blade member 14 has a boss 15 adapted to be coupled to the blade shaft 12 and blades 16 which are formed integrally with the boss 15. Each blade 16 has a beating surface 17 which is stretched in the vertical direction. A multiplicity of meshes 18 are formed in the beating surface 17. Each blade 16 is curved at its radially outer end portion toward the leading side as viewed in the direction of rotation, as at 19. Further, each blade 16 is provided with a plurality of ribs 20 projecting from the peripheral end thereof, a flange portion 21 extending from the upper edge thereof in toward the leading side as viewed in the direction of rotation, and projecting wedge portions 22, 23 projecting also toward the leading side from an intermediate portion and lower edge thereof, respectively. The lower surfaces of the flange 21 and the web 23 are tapered as at 24, 25, such that the heights of these surfaces from a reference level become large as they get remote from the main part of the blade 16, i.e., toward the leading side ends as viewed in the direction of rotation. On the other hand, the upper surface 26 of the web 22 is tapered downwardly toward the leading side as viewed in the direction of rotation. A lid 27 is detachably secured to the vessel 13. In operation, after placing a cooking material such as an egg in the vessel 13, the lid 27 is fixed to close the vessel 13 and then the motor 2 is started. The torque of the motor is transmitted to the beating blade member 14 through the transmission mechanism 6, output shaft 8 and the blade shaft 12. As a result, the blade member 14 is driven by the motor 2 and rotated at a high speed. The rotation of the beating blade member 14 causes, not only a large vortex of the whole material to stir the latter, but also a numer of local eddy currents or vortex flows in every part of the material by the action of the meshes 18. At the same time, ambient air is induced by the meshes 18, so that the material is sufficiently mixed with the air to become froth. It is therefore possible to beat or whip the cooking material in quite a short period of time without causing any fatigue of the user's hand. It has been found that the most efficient mixing and beating are performed when the ratio of the area of the meshes to the whole area of the beating surface 17 is about 60 to 90%. Provided that the width of the grid or lattice forming the meshes is 1 mm, the beat mixing and beating is attained when each mesh has an area of 0.21 cm 2. Similarly, if the grid or lattice width is 0.6 mm, the beat result is obtained when each mesh has an area of 0.1 cm 2. With above stated size of the meshes, the best result was observed when the blade assembly 14 is rotated at a speed of 600 to 1200 R.P.M. The curved end portion 19 of each blade 16 effectively forces the cooking material residing near the inner peripheral surface of the vessel 13 to flow toward the center of the vessel to enhance the stirring effect. Also, air is entrapped between the comb-teeth-like ribs 20 of each blade so as to be mixed with the cooking material. Further, the flange 21 of each blade reduces the rate of the upper flow of the cooking material which detours the blade member 14 so as to promote the mixing and beating effect provided by the meshes 18. In addition, the tapered surfaces 24, 25 and 26 in combination generates a vertical component of the flow of the cooking material to enhance the mixing and beating effect. For these reasons, it is possible to obtain a further improved efficiency of beating or whipping work. It is also advantageous that the blade member 14 has a simple construction constituted by a boss 15 integrated with blades 16. The blade member having the simplified construction can easily be attached to and detached from the blade shaft 12. The cooking material clogging the meshes 18 of the blade 16 can easily be washed away as the meshes are moved two or three times in the water. This blade member 14 is therefore quite easy to handle. A second embodiment of the invention will be described hereinunder with specific reference to FIG. 6. The beating blade member 28 of this embodiment has blades 30 integrated with a boss 29. Each blade 30 is provided with a beating surface 32 which is stretched or spread in the vertical direction. The blade 30 has a multiplicity of comb-teeth 32 extending in the vertical direction in parallel with one another. The blade member 28 is adapted to be rotated at a high speed by the motor. As a result, the blade member 28 generates a large vortex of the whole cooking material to stir the latter. At the same time, the cooking material is stirred locally at every portion thereof by the comb-teeth 32. In addition, air is entrapped between the comb-teeth 32 so as to be efficiently mixed with the cooking material. Consequently, it is possible to beat or whip the material into froth in quite a short time, without incurring the fatigue. FIG. 7 shows a third embodiment of the invention. In this third embodiment, each blade 30 is curved at its radially outer end portion where the peripheral velocity is the greatest, toward the leading side as viewed in the direction of rotation, as at 33. Consequently, the cooking material in the vicinity of the wall of the vessel is forced to flow toward the center of the vessel to enhance the stirring effect. The beating blade member 28 can have a simple construction constituted by a boss 29 and blades 30 integral with the latter, and can easily be attached and detached to and from the blade shaft. The cooking material clogging the space between adjacent comb-teeth 32 can easily be removed by moving two or three times the blade in the water. The blade member of this embodiment, therefore, can be handled quite easily. FIGS. 8 to 10 inclusive show a fourth embodiment of the invention, in which the same reference numerals are used to denote the same parts or members to those of the previously described embodiments, and the description of these parts or members are omitted. The beating blade assembly 28 of this embodiment has blades 30 in each of which formed are comb-teeth 32. The blade 30 also has radially outer end portion 33 which is curved toward the leading side as viewed in the direction of rotation. Further, a flange 34 is formed on the upper edge of each blade 30 to project forwardly therefrom, i.e. toward the leading side as viewed in the direction of the rotation. The lower surface 35 of the flange 34 is tapered upwardly toward the leading side as viewed in the direction of rotation. In operation, the curved end portion 33 causes an inward flow of the cooking material to enhance the stirring effect. Also, the flange 34 prevents the cooking material from passing above the blade member 28, so as to enhance the chance of interfere of the cooking material with the comb-teeth 32. The tapered lower surface 35 of the flange 34 causes a vertical stirring action. Consequently, the beating or shipping effect is further enhanced. FIG. 11 shows a fifth embodiment of the invention. The beating blade member 28 used in this embodiment has a multiplicity of ribs 36 projecting outwardly from radially outer end of each blade 30. In addition to the whipping effect of the comb-teeth 32 as stated above, stirring is effected locally at every part of the cooking material by the ribs 36 which are situated at the outer extremity of each blade 30 where the peripheral velocity is the greatest. In addition, air is conveniently caught between adjacent ribs 36, so that a more vigorous mixing of the air and cooking material is achieved. A sixth embodiment of the invention will be described hereinunder with specific reference to FIG. 12. The beating blade member 37 of this embodiment has a boss 38 by which it is connected to the blade shaft and blades 39 integral with the boss 38. Each blade 39 has a vertically stretched beating surface 40 constituted by a multiplicity of laterally extending slit-like bores 41 defined by a grid 42. In operation, the motor is started after a cooking material such as an egg is placed in the vessel. The beating blade member 37 is rotatively driven by the motor at a high speed. The cooking material as a whole is stirred in the form of a vortex flow. At the same time, the laterally extending slit-like bores 41 and the grid 42 in combination generate local vortex or eddy current of the material at every part of the latter so as to enhance the stirring effect. In addition, air is induced into the bores 41 so as to be efficiently mixed with the cooking material. Consequently, the cooking material and the air is sufficiently mixed to complete the beating or whipping of the cooking material in a short time without incurring fatigue. FIG. 13 shows a seventh embodiment of the invention. In this embodiment, the radially outer end portion of each blade, where the peripheral velocity is the greatest, is curved toward the leading side as viewed in the direction of rotation, as at 43. This curved end portion 43 of each blade effectively causes a radially inward component of flow of the cooking material so as to further enhance the whipping action. The beating blade member has a simple construction constituted by a boss 38 and blades 39, and can easily be attached to and detached from the blade shaft. The cooking material attaching to the bores 41 and the grid 42 can easily be removed by moving the blade two or three times in the water. The blade member of this embodiment thus can easily be handled. An eighth embodiment of the invention will be described with reference to FIGS. 14 to 16. The parts or members same as those of the embodiments described before are denoted by the same reference numerals and are not detailed here. The beating blade member 44 of this embodiment has blades 39 in each of which formed is a grid 42 which defines a plurality of slit-like bores 41. The radially outer end portion of each blade is curved as at 43 toward the leading side as viewed in the direction of rotation. A flange 45 is formed unitarily with the blade 39 at the upper edge of the latter to extend forwardly therefrom, i.e. toward the leading side as viewed in the direction of rotation. The lower surface 46 of the flange 45 is tapered upwardly toward the leading side as viewed in the direction of rotation. In operation, the curved portion 43 of each blade caused an inward flow of the cooking material to enhance the stirring effect. At the same time, the flange 45 prevents the cooking material from passing above the beating blade member 44 thereby to enhance the chance of interefere of the cooking material with the bores 41 and grids 42 of the blades 39. Consequently, the beating or whipping is more efficiently. A ninth embodiment of the invention is shown at FIG. 17. The beating blade member 47 as used in this embodiment is provided with a plurality of ribs 48 projecting in the form of comb-teeth from the radially outer end of each blade 39. In operation, in addition to the above described beating or whipping action performed by the bores 41 and grids 42, local stirring is effected at every portion of the cooking material as a whole by the ribs 48 which are disposed at the radially outer extremity of each blade 39 where the peripheral velocity is greatest. In addition, air is conveniently caught between the adjacent ribs 48 so as to permit a more vigorous mixing of the cooking material with the air. A tenth embodiment of the invention will be described with reference to FIGS. 18 and 21. The beating blade member 49 of this embodiment is provided with a boss 50 through which it is connected to the blade shaft and blades 51 which are formed integrally with the boss 50. Each blade 51 has a vertically stretched beating surface 52 in which formed are a multiplicity of meshes 53. At the same time, the blade 51 is provided with a plurality of comb-teeth 54 extending downwardly therefrom. Further, the radially outer end portion of each blade 51 is curved as at 55 toward the leading side as viewed in the direction of rotation. Further, each blade 51 is provided with a plurality of ribs 56 extending in the form of comb-teeth radially outwardly from the outer extremity thereof, and also with a flange 57 which extends toward the leading side as viewed in the direction of rotation from the upper edge thereof. The flange 57 has a lower end surface 58 which is tapered upwardly toward the leading side as viewed in the direction of rotation. In operation, the motor is started after a cooking material such as an egg is placed in the vessel. As a result, the the beating blade member 49 is rotatively driven at a high speed by the motor, so that the material as a whole is rotated to form a vortex flow to enhance the stirring. At the same time, the meshes 53 and the comb-teeth 54 imparts local stirring action at every portion of the cooking material. Further, the meshes 53 conveniently traps the air so as to permit a sufficient mixing of the cooking material and air with each other. It is, therefore, possible to beat or whip the cooking material into froth in quite a short period of time, without incurring fatigue of the user's hand. The best mixing of the cooking material with air is achieved with the ratio of opening area of the meshes 53 to the area of the entire beating surface 52 of about 60 to 90%, opening area of each mesh opening of 0.21 cm 2 (in this case, the width of grid forming the meshes is 1 mm) or 0.1 cm 2 (in this case, the width of grid forming the meshes is 0.6 mm) and a revolution speed of the beating blade member falling within the range of between 600 and 1200 R.P.M. Further, by the presence of the curved portion 54 of each blade, the cooking material residing in the vicinity of the inner peripheral surface of the vessel is forced to flow toward the center of the vessel so as to enhance the stirring effect. At the same time, air is caught between adjacent comb-like ribs 56 so as to be mixed efficiently with the cooking material. Further, the flange 57 effectively prevents the cooking material from passing above the blades 51 so as to enhance the mixing action performed by the meshes 53. The tapered lower surface 58 of the flange generates a vertical flow component of the cooking material to promote the stirring. As a result, a more efficient beating or whipping is performed. In addition, the beating blade member 49 can have a simple construction constituted by a boss 50 and blades 51 integral with the latter, and can easily be attached and detached to and from the blade shaft. The cooking material sticking to the blades 51 can easily be washed away as the blades are moved two or three times in the water. The beating blade member of this embodiment, therefore, can be handled in quite an easy manner. An eleventh embodiment of the invention will be described with reference to FIGS. 22 and 24. The beating blade member 59 of this embodiment has a boss 60 at which it is connected to the blade shaft and blades 61 formed integrally with the boss 60. The blade 61 has a vertically stretched beating surface 62 in which are formed a multiplicity of laterally extending slit-like bores 63 defined by a grid 64. The blade 61 is also provided with comb-teeth projecting downwardly from the lower edge thereof. Further, the radially outer end portion 66 of the blade 61 is curved toward the leading side as viewed in the direction of rotation. In addition, a flange 67 is formed to extend forwardly, i.e. toward the leading side as viewed in the direction of rotation, from the upper edge of the blade 61. The lower surface 68 of the flange 67 is tapered upwardly toward the leading side as viewed in the direction of rotation. In operation, a cooking material such as an egg is put into the vessel. Then, after attaching the lid, the motor 2 is started. Consequently, the beating blade member 59 is rotatively driven at a high speed to cause a vortex flow of the whole material to stir the latter. Simultaneously, the girds 64 and the comb-teeth 65 generate local vortex flows or eddy currents of the cooking material at every portion of the latter to promote the stirring. At the same time, the bores 63 positively introduce the air to promote a sufficient mixing of the cooking material with air. It is therefore possible to beat or whip the cooking material into froth in quite a short time, without incurring fatigue. Further, the curved portion 66 of each blade 61 acts to cause the cooking material staying in the vicinity of the inner peripheral surface of the vessel to flow toward the center of the latter, so as to further promote the mixing and stirring of the cooking material. In addition, the flange 66 acts to reduce the detouring flow of the cooking material above the blades 61 so as to enhance the mixing effect performed by the blades 61. The tapered lower surface 68 of the flange 66 conveniently generates a vertical component of flow of the cooking material to promote the stirring. The beating or whipping is therefore rendered more efficient. It is also possible to provide a plurality of ribs 69 projecting radially outwardly from the peripheral edge of each blade 61 in comb-teeth-like form, as shown in FIG. 24. By so doing, a more effective stirring action is ensured, because these ribs 69 promote the stirring action and because the air is caught between adjacent ribs 69 to provide a more efficient mixing of the air with the cooking material. The beating blade member 59 can have a simple construction consisting of a boss 60 and blades 61, and can easily be attached and detached to and from the blade shaft. The cooking material sticking to the blades 61 can be removed easily by moving the blades 61 two or three times in the water. The beating blade 59 thus can be handled in quite an easy manner. A twelfth embodiment of the invention will be described with reference to FIGS. 25 and 26. The beating blade member 70 of this embodiment has a boss 71 at which it is attached to the blade shaft and stirring blade portions 72. Each stirring blade portion 72 is notched at its central portion to provide recessed portion 73 so as to reduce the resistance by which the blade portions are encountered during the stirring of the cooking material put in the vessel. More specifically, each stirring blade portion 72 is constituted by a laterally extending horizontal portion 74 and a vertical portion 75 extending upward from the outer end of the latter. A plurality of small rods 76 made of nylon or the like material and hence exhibiting a small resistance to the cooking material are provided on the outer extremity of the stirring blade portion 72. At the same time, a plurality of small rods 78 are formed on the stirring surface 77 of each stirring blade portion 72, so as to extend obliquely downwardly at an inclination of 45° to the horizontal plane. The root portions of these small rods 78 are staggered by half pitch from the root portions of the small rods 76. In operation, a cooking material such as an egg is put in the vessel, and the motor is started after the attaching of the lid. The torque of the motor is transmitted to the beating blade member 70. Since the beating blade member 70 is provided with recessed portions 73 in its stirring blade portion 72, the beating blade member 70 can be rotated at a high speed, because of the reduced resistance. As a result, the small rods 76,78 are rotated at a high speed to splash about the cooking material to stir the latter at a high speed, while inducing air to promote the mixing of the cooking material with air. Consequently, the cooking material is whipped into froth effectively in quite a short period of time. The small rods 76 disposed at the peripheral portion of the beating blade member effectively promote the beating or whipping because of their high peripheral velocity during rotation. The other small rods 78 provided on the beating surface effectively stir the cooking material because they are formed to project obliquely from the beating surface. In addition, since the small rods 76,77 are staggered by half pitch, the splashing stirring action is rendered more vigorous. Since the beating blade member 70 is rotated only in one direction, the beating or whipping can be made without any specific skill, even when the beating blade member 70 is rotated manually. Further, the beating blade member 70 can have a simple construction, because it requires only one boss 71, and can easily be attached and detached to and from the blade shaft. The cooking material sticking to the beating blade portion 72 can easily be washed away by moving the same two or three times in the water. FIGS. 27 to 29 in combination show a thirteenth embodiment of the invention. The parts or members the same as those of the previously described embodiment are denoted by the same reference numerals and are not detailed here. In this embodiment, tabular members 79,80 of substantial areas are fixedly attached to the outer edge and lower edge of the stirring blade portion 72. In addition, a plurality of rows of small rods 76 are formed on these tubular members 79,80. These rows are staggered by a half pitch. Thus, the beating blade member 81 of this embodiment can have a larger number of the small rods 76 and hence can provide a further efficient beating or whipping action. Finally, a fourteenth embodiment of the invention will be described with reference to FIGS. 30 to 36. At the center of the bottom of a vessel 82, formed is a cylinder 84 from which projected is a spindle 83 operatively connected to a reduction gear. A boss 85 having a cylindrical form with closed upper end is fitted around the cylinder 84. The boss carries blades 86 formed unitarily therewith. Needless to say, it is possible to fabricate the boss 85 and the blades 86 as separate bodies and to assemble them into a beating blade member. The boss 85 is attached to the spindle 83 for free axial displacement but is prevented from rotating relatively to the latter. Each blade 86 extends radially outwardly from the boss 85 and has a beating surface 87. The beating surface 87 is curved toward the leading side as viewed in the direction of rotation. Meshes 88 are formed in each beating surface 87. The distance a between the radially outer extremity of the beating surface 87 and the inner peripheral surface of the vessel 82 is selected to be 1 mm. At the peripheral portion of the beating surface 87, is formed an inclined surface 89 which is inclined toward the leading side of rotation by an angle of 45°. The outer extremity of this inclined surface 89 terminates in an edge 90. An upper and a lower flanges 91,92 are formed on each beating surface 87 so as to oppose to each other. The upper flange 91 has a projection 93 which extends on the extension of the edge 90 to oppose to the inner peripheral surface of the vessel 82 with a small distance a of 1 mm, and a plurality of comb-teeth-like ribs 94. At least the innermost one of the ribs 94 has a tapered surface 95 which is tapered radially outwardly toward the leading side as viewed in the direction of rotation at an angle of 45°. The lower flange 92 is provided with a plurality of comb-like ribs 96. The ratio of the breadth b of each rib 96 to the distance c between adjacent rib 96 is given by the equation of: b:c=1:3. The distance d between the rib 96 and the bottom surface of the vessel 82 is 1 mm. In operation, for preparing a mayonnaise for example, the material is put into the vessel 82, and the motor is started to rotatively drive the boss 85 together with the blades 86. As a result, the beating surface 87 stirs the material as a whole in the circumferential direction. During this stirring action, the meshes 88 generates local vortex or eddy currents of the material at every portion of the latter and induce the ambient air to effectively perform the beating or whipping. The material tends to stick to the inner peripheral surface of the vessel 82, particularly when the viscosity is high. The sticking material, however, is effectively scraped off by the edge 90 of each blade which are positioned in the close proximity of the inner peripheral surface of the vessel. The scraped material is then moved toward the center of the vessel 82, and is then directed radially outwardly. When the amount of the material placed in the vessel is not so large, the material is raised above the radially outer end portion of the blades 86, as shown in FIG. 35. This raised material, however, is effectively stirred by the projection 93 which is located at a distance of 1 mm from the inner peripheral surface of the vessel 82. It will be seen that an effective beating or whipping can be performed even with a small amount of material. At the same time, the ribs 96 formed on the down surface of the flange 92 acts to further promote the beating or whipping. The beating or whipping will be further rendered vigorous by arranging such that the ribs 96 on one of the blades is staggered in the radial direction from the ribs 96 on the other blade, i.e. such that the ribs at both sides of the boss 88 are staggered in the radial direction. As the amount of the material placed in the vessel 82 is increased, the swell of the material above the beating blade member 89 is gradually moved toward the center of the vessel. This swell is effectively stirred by the ribs 94. Particularly, the air and the material colliding with the inclined surfaces 95 of the ribs 94 are introduced inwardly and effectively mixed with each other. It is therefore possible to beat and whip the material effectively into froth, even with a large amount of the material. Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein. | Summary: A beating blade member for beating or whipping cooking material such as egg into froth. The blade member has blades each of which has a vertically stretched and radially extending beating surface in which formed are a multiplicity of meshes. Thanks to the provision of these meshes, it is possible to obtain fine contact of the material with the blade to promote the stirring and beating. Also, these meshes effectively catch the ambient air to permit a better mixing of the material with the air. At the same time, the material is guided to flow radially inwardly, as well as in the vertical direction, so as to ensure the interference of the material with the meshes of the blades. Consequently, the material is beaten and whipped into froth in quite a short period of time. | 8,260 | 179 | big_patent | en |
Summarize: Overview For most of the first four decades after the country was founded in 1948, South Korea was ruledby authoritarian governments. Ever since the mid-1980s, when widespread anti-government protestsforced the country's military rulers to enact sweeping democratic reforms, democratic institutionsand traditions have deepened in South Korea. In 1997, long-time dissident and opposition politician Kim Dae Jung (commonly referred to as "DJ") was elected to the presidency, the first time anopposition party had prevailed in a South Korean presidential election. By law, Korean presidentscan only serve one five-year term. In December 2002, Kim was succeeded by a member of his party, Roh (pronounced "noh") Moo-hyun, a self-educated former human rights lawyer whoemergedfrom relative obscurity to defeat establishment candidates in both the primary and general elections. Roh campaigned on a platform of reform -- reform of Korean politics, economic policymaking, andU.S.-ROK relations -- to narrowly defeat conservative candidate Lee Hoi Chang in the generalelection. Roh captured 48.9% of the vote to Lee's 46.6%. Prior to assuming the Presidency, Roh's only previous government experience was an eight-month term as Minister of Maritime Affairs and Fisheries in 2000 and 2001, and two stints asa lawmaker in the National Assembly, from 1988-1992 and 1998-2000. During the 1990s, Roh losttwo legislative elections and one bid for the mayoralty of his hometown, Pusan. Ironically, duringthe presidential campaign, the circumstances surrounding these defeats contributed to Roh's imageas a principled reformer. Noteworthy Aspects of the 2002 Presidential Election. The 2002 election was notable for several inter-related reasons. First, itexposed the deep generational divide among South Koreans. Exit polls showed that nearly 60% ofpeople in their 20s and 30s voted for Mr Roh, compared with less than 40% of people in their 50sand 60s. It is perhaps not coincidental that those supporting Lee directly experienced the KoreanWar (1950-53) and its immediate aftermath. Roh, 56, himself represents a generational shift. Eleven years younger than Lee, he also is the first South Korean president who was not an established political figure when the country began toemerge from authoritarian rule in the late 1980s. Second, Roh's victory was due in part to his call for South Korea to become a more "equal" partner in the U.S.-ROK alliance and to his criticism of U.S. policies on the Korean peninsula. Rohpledged to largely continue Kim Dae Jung's "sunshine policy" of engaging North Korea. Althoughthese views resonated most strongly among younger generations of South Koreans, they have enteredthe mainstream of Korean society, as shown by the massive demonstrations in late 2002 protestingthe acquittal of two U.S. servicemen who were operating a military vehicle when it killed twoKorean schoolgirls. Third, the election and the anti-American demonstrations highlighted the growing influence and sophistication of Korean civil society groups, which now have a significant voice in determiningpolicy outcomes in Seoul. Many of these organizations garnered support for Roh during the electioncampaign and organized the anti-U.S. protests and the "Red Devil" rallies to cheer South Korea'snational team on to the semifinal during the 2002 Soccer World Cup. The groups' goals and means-- which tend to rely heavily upon networking through the Internet -- have been particularlyeffective in encouraging younger Koreans to become politically active. Higher-than-expectedturnout of younger voters benefitted Roh, as overall turnout in the 2002 election was a record lowof 70%, compared with 80% in the 1997 election. A Country Divided. The challenge facing Roh, who was inaugurated on February 25, 2003, is how to carry out his reform agenda despite the deepdivisions in South Korea. The country is split not only generationally, but also regionally. TheNational Assembly is controlled by the opposition Grand National Party (GNP). And Roh's ownparty, the Millennium Democratic Party (MDP) is also divided between his supporters and a largefaction of party elders that nearly succeeded in ousting Roh as the MDP's presidential candidateseveral weeks before the election. Roh's task will be complicated by the National Assembly elections scheduled to take place in April 2004. These are likely to exacerbate the zero-sum tendency in South Korean politics, in whichruling and opposition parties rarely cooperate. Furthermore, both the ruling MDP and oppositionGNP are under significant internal stress, and there is speculation that there will be a major politicalrealignment before the next legislative election. South Korea's Political System A Weak Legislature. Nominally, power in SouthKorea is shared by the President, who is elected to one five-year term, and the 273-memberunicameral National Assembly. National Assembly members are elected to 4-year terms. Of these,227 members represent single-member constituencies. The remaining 46 are "at large" membersselected on the basis of proportional voting. As mentioned above, the next legislative elections willbe held in April 2004. Since the Assembly's powers were enhanced in the 1987 constitution, it has at times altered the political climate by providing opposition parties with a forum to criticize, inspect, and attempt toembarrass the executive branch. Indeed, one of the Assembly's major accomplishments since 1987has been to institutionalize its oversight of the executive; executive branch officials regularly appearbefore committees, helping to make South Korean policy-making more transparent. Also, from timeto time, opposition parties have used Assembly proceedings to successfully stymie presidentialinitiatives, usually by boycotting legislative sessions. In reality, however, the President continues to be the dominant force in South Korean policymaking, as formal and informal limitations prevent the National Assembly from initiatingmajor pieces of legislation. For instance, the Korean constitution grants the Assembly the poweronly to cut funds from the President's budget, not to propose any increases or alter the executive'sbudgetary allocations. (Article 57) Also, the Assembly must act on the budget not later than thirtydays before the start of the new fiscal year on January 1. (Article 54) South Korean legislators suffer from numerous other limitations. They have little in the way of legislative support agencies, and the typical Seoul legislative office is staffed by three salaried,full-time workers. Even the Prime Minister, who has little power, is nominated by the President. The fact that parties in the Assembly repeatedly have resorted to the brinkmanship tactic of theboycott reflects the legislative branch's weakness. Institution-Building by the National Assembly. Roh's election has accelerated moves by the opposition Grand National Party (GNP), which controls theNational Assembly, to increase the National Assembly's resources through the creation ofinstitutions modeled after the U.S. Congressional Budget Office, the Congressional ResearchService, and/or the General Accounting Office. The Assembly's Other Powers. A bill in the National Assembly may be introduced by members or by the executive branch. If passed, a bill is sent to theexecutive for presidential promulgation within 15 days. The President may veto the bill and sendit back to the National Assembly for reconsideration. If the assembly overrides the veto with theconcurrence of two-thirds or more of members present, the bill will become law. With respect tothe judiciary, the consent of the National Assembly is also required for the presidential appointmentof all Supreme Court justices. Also required is the ratification by the Assembly of treaties andagreements as well as international acts or conventions to which South Korea is a signatory. Thedeclaration of war, the dispatch of troops abroad, or the stationing of foreign troops in South Koreanterritory requires the consent of the Assembly as well. PDFversion Political Parties. South Korean political partiesform, disband, and merge regularly, in part because they tend to be regionally-based and centeredaround charismatic personalities rather than substantive issues. It is not uncommon for members ofthe National Assembly to jump from one party to another, a practice that has led to considerabledisaffection among the South Korean electorate. During the 2002 presidential election, RohMoo-hyun benefitted from his image of a principled politician who had suffered personally becauseof his resolution not to participate in these political machinations. In 1990, Roh chose not to followhis mentor and long-time opposition leader, Kim Young Sam, into an alliance with the then-rulingparty, a decision that contributed to Roh's defeats in the 1992 National Assembly election and the1995 Pusan mayoralty election. In 1992, Kim won the presidential election. Presently, there are three major political parties in South Korea: President Roh Moo-hyun's center-left ruling Millennium Democratic Party ( MDP, formerly the National Congress for NewPolitics), which has 101 seats in the National Assembly (137 seats constitutes a parliamentarymajority); the generally conservative opposition Grand National Party ( GNP ), which at 153 seatshasa majority in the Assembly; and the smaller (14 seats) conservative United Liberal Democrats(ULD), which is led by the veteran politician Kim Jong-pil and has declined dramatically in size andinfluence in recent years. In April 2003, the GNP further increased its majority by winning 2 of 3bi-elections that were notable for a turnout of below 30%, the lowest in nearly 40 years. There is speculation that there may be a major political realignment before the April 2004 National Assembly elections. The MDP and GNP both are undergoing unsettled times. The GNP,which suffered from defections early in the presidential campaign, is still recovering from Lee HoiChang's defeat. The divisions are even more acute in the MDP, where Roh's base of support withinthe ruling party is confined to a relatively small group of younger reformers. During the presidentialelection, the party's "establishment" figures favored another candidate who Roh defeated in theMDP's primaries -- the country's first-ever -- in the winter of 2002. Later in the presidentialcampaign, opposition to Roh inside the MDP grew to such an extent that several party leaderspublicly talked of disbanding and forming a new party led by another candidate. Some politicalanalysts speculate that Roh may seek to gather into a new party reformist politicians from the MDP,GNP, and smaller parties. Regionalism. South Korean politics is highly regionalized. The MDP's base is in the southwestern Cholla region (also known as Honam), whereRoh won 93% of the vote in the 2002 presidential election. (See Figure 2) In the last nation-wideNational Assembly elections, held in April 2000, no GNP candidate won any of Cholla's 29 seats. Conversely, GNP candidate Lee Hoi Chang won 71% of the vote in the southeastern Kyongsang region (also known as Yongnam), the GNP's stronghold. In the 2000 National Assembly elections,the GNP won 64 of the 65 constituencies in Kyongsang. Before Kim Dae Jung's victory in 1997,South Korea's military governments gave preferential treatment to the Kyongsang region, leadingCholla residents to accuse the government of discrimination. The greater Seoul area, which is thehome of nearly half of the South Korean electorate, has emerged as the swing vote. There has beendiscussion of combating regionalism by adopting multiple-seat constituencies in the NationalAssembly. Corruption. (1) NGOs. Since the end of military rule, non-governmental organizations (NGOs) have increased dramatically in South Korea, particularlyin the past five years. The groups exist on both the local and national levels, range widely in size,and focus on a wide array of issues, including the environment, government and corporatecorruption, disability rights, women's rights, crimes committed by U.S. forces in Korea, revising theU.S.-ROK Status of Forces Agreement (SOFA), and returning land used by U.S. forces. Manyinternational NGOs have set up local chapters in Seoul. In contrast to the student-led, class-basedgroups of the 1980s that spent much of their time organizing militant anti-government and anti-U.S.protests, an increasing number of NGOs have hiring permanent staff, fundraisers, and researchoffices. (2) As they have begun to professionalize theiroperations, they seem to have begun to havea greater impact on South Korean domestic and foreign policy. South Korean NGOs have been particularly adept at forming loose, temporary coalitions with one another to organize large-scale protests on a particular issue. Many of the most successfulexamples of citizen activism in South Korea involved the formation of umbrella organizations thatpool the resources of member groups. The country's rapid adoption of the Internet -- South Koreahas one of the world's highest rates of Internet usage -- has facilitated such networking by enablinggroups to quickly establish linkages, coordinate activities, and spread the word about protestactivities. (3) The anti-American protests in 2002 werea case in point. Decentralization. The growing influence of Korean civil society groups has been possible in part because of the growing decentralization ofpower in South Korea. In the early and mid-1990s, new laws were passed creating local assembliesand establishing popular election of local officials for the first time since the 1950s. Increased localautonomy has encouraged political consciousness and activism, as South Koreans have come toexpect local and national elected leaders to be more responsive and accountable to theirconstituents. (4) Despite these changes, Seoul remainsthe locus of political power in South Korea, inpart because local governments have little authority to impose their own taxes. (5) President RohMoo-hyun has vowed to further decentralize political power giving more autonomy to the provincesand by transferring certain executive offices to locations outside of Seoul. South Korea's Media. During the country's period of military rule, South Korea's media operated under tight censorship rules. Additionally,the government rewarded favorable coverage by exempting Korean newspapers from intrusivescrutiny into the companies' internal operations. In recent years, however, tensions between thegovernment and South Korea's print media have surfaced, and appear to have worsened under thecurrent Roh administration. South Korea has nearly two dozen major newspaper and broadcast companies. Korea's three leading mainstream newspapers, the Chosun Ilbo, Donga Ilbo, and the JoongAngIlbo, control about70% of the market. All claim circulation figures of between 1.8 and 2.1 million and haveright-of-center political leanings. The leading left-of-center daily, Hankyoreh, has an estimatedreadership of 400,000. (6) In recent years, manyyounger South Koreans have turned to internet newssources such as OhMyNews. Most Korean newspapers are affiliates of and/or highly dependent on the advertising revenues from the country's chaebol conglomerates, giving many an allegedly predisposed hostility to the chaebol reforms pushed by former President Kim Dae Jung and continued by President Roh. Traditionally, mainstream newspapers have been opaque operations that have benefitted theircorporate patronage. Circulation figures are not independently audited, journalists are known toaccept pay-offs from companies and government agencies to supplement low wages, and for yearsnewspapers were exempt from tax audits. The government often has been reluctant to criticize themedia because of fears that owners would retaliate by publishing embarrassing stories. Koreannewspapers' political biases frequently are reflected in their news coverage, and articles often arebased upon rumors or hearsay rather than actual evidence. (7) In 2001, President Kim Dae Jung launched a highly public tax probe of the country's leading dailies that was criticized for targeting those papers that were most critical of the government. During the 2002 campaign, President Roh frequently complained about a "conservative" bias in thepress and made greater openness in the media an element of his reform platform. Since hisinauguration, Roh has acted to contain "unfair" ways newspapers "distort" government policies byreplacing restrictive press clubs with open press briefings and by requiring government agencies toprepare daily summaries that classify newspaper articles into one of five categories: "positive,straight and simple, constructive criticism, malicious criticism, and error." The reports are then tobe sent to the Blue House, the President's office. Criticism over these moves contributed to theresignation of Roh's appointee as president of the state-run Korean Broadcasting System (KBS). Roh has also moved to grant greater access to Korea's internet publications that are widely used byyounger readers and many of which backed Roh during the election campaign. (8) Current Political Situation and Analysis Roh Moo-hyun won the South Korean Presidency by adopting a mantle of reform, one thatcovers three areas: Korea's relations with the United States, Korea's economic policy, and Koreanpolitics. Additionally, Roh has pledged to continue the essence of Kim Dae Jung's sunshine policytoward North Korea, which he has renamed the "peace and prosperity" policy. "Anti-Americanism". Anti-American sentiments have long been present in South Korean society, particularly during the 1970s and 1980s. After theend of military rule they were largely confined to highly ideological groups, particularly studentorganizations, that generally operated on the political margins in Korea. In the late 1990s, however,criticisms of United States policy moved into the mainstream, a move that also has madeanti-Americanism less ideological and more issue-specific. The criticisms range widely and includeaccusations that the Bush Administration is not listening to South Koreans in general, that the BushAdministration is blocking rapprochement between North and South Korea, that U.S. forces in SouthKorea are not sufficiently accountable for crimes they commit in South Korea, that the United Statesis covering up alleged atrocities committed during the Korean War, and that the South Koreangovernment too often caters to U.S. interests. (9) Underlying these sentiments is the declining senseof threat that South Koreans feel from North Korea. This trend was evident in the late 1990s, andhas deepened since the North-South Korean summit in June 2000. (10) Two events in 2002 caused thecritiques to coalesce into massive anti-U.S. demonstrations: President Bush's inclusion of NorthKorea in his "axis of evil" countries, and the June 2002 killing of two Korean schoolgirls by a U.S.military vehicle. Critics of the United States tend to be younger than 50, and appear to fall into three broad groups: 1) radical leftists, many of whom ideologically reject the U.S.-ROK alliance and some ofwhom support North Korea; 2) nationalists, who resent perceived intrusions into South Korea'ssovereignty by the United States (most prominently, the U.S.-South Korea status of forcesagreement) but who do not necessarily oppose the alliance per se ; and 3) individuals who supportthe alliance but oppose U.S. policy on specific issues, such as alleged crimes committed by U.S.servicemen. This latter group appears to be in the majority among current anti-American activists. (11) Some analysts have pointed out that a failure to address the concerns of the latter group couldexacerbate the problem by causing these individuals' concerns to morph into a nationalistic orideological-based opposition to the U.S.-South Korean alliance. Opinion polls taken over the past few years generally have found that large majorities of respondents favor a partial or total withdrawal of U.S. troops from South Korea, though mostholding this position say they favor a drawdown unless there are improvements in North-SouthKorean relations; few favor an outright withdrawal. The Bush Administration's initiatives to actupon longstanding, but never enacted, plans to reduce or redeploy U.S. forces, including an April2003 pledge to move from the Yongsan base in Seoul, appear to have calmed the anti-basemovement somewhat. The Roh government has asked the U.S. not to undertake these moves untilthe nuclear issue with North Korea is settled. Relations with the United States under Roh. During the presidential campaign, Roh tapped into anti-American sentiments in several ways. Hecriticized the Bush Administration for not negotiating with North Korea. He called for"modernizing" the U.S.-ROK alliance to make South Korea a more equal partner in the relationship. He demanded a renegotiation of the U.S.-South Korea Status of Forces Agreement (SOFA). And,Roh bucked South Korean tradition by not traveling to the United States during the campaign. Indeed, Roh's May 14, 2003 summit with President Bush in Washington, DC will be his first tripto the United States. In the late 1980s, Roh called for a complete withdrawal of U.S. forces after ahoped-for reunification with the North, a stance he backed away from during the campaign. Since winning the election, Roh has sought to reduce bilateral tensions by moderating the tone of many his statements regarding U.S.-South Korean relations. Additionally, in March, he riskedalienating many of his supporters by publicly supporting the U.S.-led war against Iraq and promisingto send 700 engineering and medical troops to Iraq. Polls show that about 80% of South Koreansopposed the invasion of Iraq. While Roh has expressed reservations about the war, he has justifiedthe move as necessary to maintain South Korea's ability to influence U.S. policy toward NorthKorea. The move prompted large anti-war demonstrations in South Korea, leading the South KoreanNational Assembly to delay a vote on the deployment three times. In response, Roh appeared beforethe National Assembly -- a rare event in South Korea -- to personally appeal for the measure. Thedeployment was approved on April 2, 2003, thanks to support from the opposition GNP, which haslong supported close alliance relations with the United States. Relations with North Korea. Underlying the wave of America bashing in South Korea is the declining sense of threat most South Koreans feel fromNorth Korea. During the presidential campaign, Roh called for a continuation of Kim Dae Jung'ssunshine policy of engaging North Korea, albeit under a different name and with broader domesticsupport. He frequently stated that "South and North Korea are the two main actors in inter-Koreanrelations." Additionally, he often criticized the Bush Administration's refusal to negotiate withPyongyang, and on the eve of the election stated that South Korea should try to play the role of asemi-neutral mediator over the nuclear dispute between the two countries. Roh has called for the North to dismantle its nuclear weapons program, though he has opposed the Bush Administration's desire to use economic sanctions to achieve this end. Instead, he hascalled for a peaceful resolution of the problem through dialogue. In his inaugural address, Roh put forth the vague outlines for a "policy of peace and prosperity" on the Korean Peninsula, one that he said will include a greater transparency than was true of thesunshine policy. This last item was a reference to $500 million in secret payments the Hyundaicorporation made to North Korea shortly before the historic North-South Korean summit in June2000. In March 2003, Roh announced he would not oppose the formation of an independentprosecutor to investigate charges that the Kim Dae Jung administration reimbursed Hyundai formuch of the payments. The prosecutor's office reportedly began its probe in April 2003. Although some in Washington, DC, viewed Roh's main opponent, Lee Hoi Chang, as a staunch critic of the sunshine policy, others believed that if elected, Lee would not have ushered in awholesale change in South Korea's policy toward the North. Though Lee called for a greater"reciprocity" in North-South relations, the main thrust of his criticisms were about implementationrather than overall direction. Lee proposed that further South Korean aid should be made conditionalupon reductions in the North's military threat, a linkage that the Kim government did not make, andthat the Roh government has only implied without making explicit. Lee also said that no new aidshould be provided until North Korea dismantles its nuclear weapons program. Economic Policy. President Roh has stated that market-oriented reforms are a key to achieving his goal of turning South Korea into "an internationallogistics and financial hub in Northeast Asia." During the campaign, Roh pledged to continue KimDae Jung's economic reforms, to force Korea's huge conglomerates ( chaebol ) to become moretransparent and to redress Korea's income gap, which has widened since the country's 1997 financialcrisis. Since his inauguration in February, Roh has pushed his chaebol reforms aggressively. Hisgovernment has encouraged the investigation and prosecution of fraudulent accounting of SKGlobal, the trading arm of the third-largest chaebol, SK. His administration also is discussingproposals to induce the chaebol to form holding companies that would reduce the power of thefounding families who currently dominate many conglomerates and reduce the practice ofcross-shareholding among individual companies within each conglomerate. Although some chaebol leaders have adopted these practices on their own, others have stated their opposition to thegovernment's proposals. Roh's ability to push through his reforms is expected to be made more difficult by the country's current economic slowdown. The Bank of Korea recently revised its economic growth forecast for2003 from 5.7% to 4.1%, due to rising tensions with North Korea, rising oil prices, decliningexports, the repercussions of the SK Global scandal, and a slump in consumer confidence. Additionally, pursuing economic reforms aggressively could require Roh to take steps that couldantagonize South Korean organized labor, which is one of the few big and coherent interest groupswith which Roh has close ties. (12) Political Reforms. By allowing the National Assembly to appoint a prosecutor to investigate Hyundai's secret payments to North Korea, Roh wasseeking to fulfill three campaign goals: make North-South relations more transparent, reducecorruption, and increase cooperation with the opposition GNP. Tactical, bipartisan alliances onindividual issues is a rarity in South Korea, where politics tend to have a zero-sum quality. TheGNP's control of the legislature, a situation that Kim Dae Jung did not face until the GNP tookcontrol in his third year in office, makes it imperative that Roh periodically work with the opposition. However, such political pragmatism risks alienating his base in the MDP. The fast-looming NationalAssembly elections, to be held in April 2004, are likely to reduce the likelihood of Roh and the GNPcooperating. Implications for the United States The U.S.-ROK Alliance Cannot be Taken for Granted The deepening of democracy and related support for populist and nationalistic policies in South Korea is likely to make U.S.-ROK alliance relations more difficult to manage and reduces U.S.options in dealing with North Korea's nuclear and missile programs. A policy of confronting Pyongyang -- for instance, by asking for the United Nations to impose sanctions -- almost certainly would require at least the tacit cooperation of Seoul to be successful. A preemptive attack on North Korea's known nuclear facilities, as was contemplated by the ClintonAdministration in 1994, would likely be strongly opposed by the Roh government and the SouthKorean people. The recent surge in anti-American sentiment also mean that a failure to obtain SouthKorea's cooperation could lead to a serious strain, if not a rupture, in the fifty year old U.S.-ROKalliance. Yet suspicions of the Bush Administration's policy toward North Korea among manySouth Koreans arguably will make it politically costly for President Roh to support a moreconfrontational approach if the United States decides to pursue this course, particularly during a yearwhen Roh's party is trying to capture the National Assembly. On the other hand, Roh's election could present the Bush Administration with an opportunity. If Roh is persuaded to follow the United States' lead, his outspoken advocacy of engaging North Korea may position him to sell a more aggressive North Korea policyto the South Korean public, which remains divided about how best to deal with North Korea. Asrecently as the summer of 2002 -- before the anti-American protests grew in size -- domesticdisenchantment with Kim Dae Jung's sunshine policy appeared sufficiently strong that Roh beganto distance himself from the approach. Anti-American Sentiment Is Likely to Continue NGO activity in South Korea tends to have an exaggerated peak-and-valley nature that appears to be a direct result of what one U.S. scholar of Korean politics has called a "loose, coalition styleof civic activism." Typically, a high-profile event results in a few groups taking the lead in puttingtogether a coalition of disparate NGOs, resulting in large-scale protests. As issues lose steam, theactivism fades dramatically because each member organization has its own agendas that may havenothing to do with the anti-American issue at hand. (13) Thus, although the recent anti-American demonstrations have largely vanished from South Korea's streets, this does not necessarily mean that anti-American sentiments have died down. Itmay only mean that the feelings are lying dormant until either another event triggers a further roundof protests or the fundamental issues of concern are addressed. The recent announcements by theU.S. Department of Defense that it plans to realign the structure of U.S. forces in South Korea mayrepresent a fundamental change that could address some of the concerns that have generatedanti-American attitudes. A Need for Greater Public Outreach? Roh Moo-hyun's election demonstrated the degree to which Korean non-governmental organizations now are able to influence South Korean politics and policy. This development placesconstraints upon the Seoul government, which is under pressure from its own citizens to be moreaccountable and transparent than ever before. In turn, this perhaps points to a need for U.S. publicdiplomacy to focus on reaching out to Korean interest groups and new online news sources. At onetime, particularly at the height of anti-American activity in the 1980s, the United States had a largepublic affairs presence across South Korea. However, all of the USIS centers in South Korea havebeen permanently closed for security reasons, replaced by Internet libraries and increased publicaffairs staff in Seoul. Additionally, there are few if any cultural exchanges conducted with SouthKorea. For decades, South Korea foreign policy was dominated by a set of elites who generally came from the same region, attended the same schools and universities, and tended to have a similarworld-view that revolved around maintaining a strong U.S.-ROK alliance. This monopoly on powerwas broken somewhat in the 1997 election of former opposition leader Kim Dae Jung. Roh'selection demonstrated that the South Korean polity has shifted further, bringing to power a new setof individuals who -- like Roh himself -- on the whole are younger, did not necessarily attend eliteschools, and are more apt to criticize the United States, particularly on matters relating to NorthKorea and to the U.S. military presence on the Korean Peninsula. Indeed, many of these individualshave complained in the past that they have been ignored by the United States, raising the point thatperhaps the U.S. government should make more concerted efforts to reach out to these groups, whichoften do not rely upon the mainstream media for news and analysis. For their part, many U.S.policymakers are worried that the number of defenders of the United States is decreasing, outsideof the conservative South Korean elite. As reported out of the House International Relations Committee on May 8, 2003, the House version of The Foreign Relations Authorization Act for Fiscal Years 2004 and 2005( H.R. 1950 ), contains four sections directed at U.S. public diplomacy in Korea,including sections that: a) would create a new, $750,000 per year program of summer institutes inthe U.S. for Korean students; b) would increase funding for the Fulbright English Teaching AssistantProgram in Korea, which sends United States citizens to serve as English language teachingassistants at Korean colleges and high schools; c) convey the sense of the Congress that theU.S.-Korea Fulbright program should try to expand beyond traditional Korean elites and eliteinstitutions; and d) convey the sense of the Congress that the Secretary of State should try toestablish a diplomatic presence in the city of Pusan. (14) | Summary: In December 2002, South Koreans elected Roh Moo-hyun, a little-known, self-educated lawyer, as their president. The left-of-center Roh narrowly defeated the conservative candidate, Lee HoiChang. Roh ran on a platform of reform, pledging to make South Korean politics more transparentand accountable, to make the economy more equitable, and to make South Korea a more equalpartner in its alliance with the United States. During the campaign, Roh pledged to continue hispredecessor, Kim Dae Jung's, "sunshine policy" of engaging North Korea, and harshly criticized theBush Administration's approach to Pyongyang. The 2002 election was notable for several inter-related reasons. First, it exposed the deep generational divide among South Koreans. Roh was favored by voters under the age of 45, whoemerged during the election as an anti-status quo force. In contrast, Lee easily won the votes ofthose over 45. Second, Roh's victory was due in part to his criticisms of the United States, and hebenefitted from the massive demonstrations in late 2002 protesting the acquittal of two U.S.servicemen who were operating a military vehicle when it killed two Korean schoolgirls. Third, theelection and the anti-American demonstrations highlighted the growing influence and sophisticationof Korean civil society groups, which now have a significant voice in determining policy outcomesin Seoul. These shifts in the South Korean polity, particularly the rise in anti-Americanism, confront the Bush Administration with a policy dilemma: how to manage the U.S.-ROK alliance while pursuinga more confrontational approach toward North Korea than that favored by many, if not most, SouthKoreans. Institutionally, the South Korean presidency has few checks on its power. While the unicameral National Assembly's influence has been slowly rising since South Korea became a democracy in1987, it is hampered by formal and informal limitations on its power. The National Assembly iscontrolled by the opposition, right-of-center Grand National Party (GNP). The second-largestgrouping is President Roh's party, the Millennium Democratic Party (MDP). Both major parties areunder significant internal stress, and there is speculation that they will split and be reconstitutedbefore the next quadrennial legislative election, to be held in April 2004. This report will be updated periodically. | 7,543 | 538 | gov_report | en |
Summarize: BACKGROUND OF THE INVENTION Various types of exercisers have been heretofore designed whereby a first limb of a person carrying out an exercise may be opposed by a second limb of that person. In addition, further exercisers have been provided whereby each limb to be exercised may be opposed by a predetermined amount of weight. Examples of exercisers of these types are disclosed in U.S. Pat. Nos. 2,716,027, 3,117,782, 3,162,441, 3,834,694 and 3,858,874. However, most previously known exercising machines are not capable of allowing one limb of a person exercising to function in opposition to another limb with both limbs in opposition opposing a variable weight load in one direction of movement. By such construction controlled exercising of a person's limbs may be carried out in an efficient manner and in a manner whereby the muscles being exercised may have the resistance loading thereon gradually increased throughout an extended period of muscle development through exercising. BRIEF DESCRIPTION OF THE INVENTION The exerciser of the instant invention is constructed in a manner whereby one limb may be exercised in opposition to a second limb of the person using the exerciser and the two limbs being exercised may be opposed, in one direction of movement thereof, by adjustable weight loading of the exercise apparatus. Of course, the exercise apparatus may also be utilized to exercise one limb or other body portion at a time, but the most efficient use of the exerciser involves the exercise of two limbs, simultaneously, in opposition to each other. The main object of this invention is to provide an exercise apparatus which may be used in a controlled manner to simultaneously exercise a pair of limbs of the user of the exercise apparatus and for exercising two pairs of limbs simultaneously, if desired. Another object of this invention is to provide an exerciser which will enable the two limbs being exercised to be exercised in opposition to each other. Yet another important object of this invention is to provide an exerciser including structure whereby movement of the two limbs being exercised may be opposed by adjustable weight loading in one direction of movement thereof. Another very important object of this invention is to provide an exerciser constructed in a manner whereby it may be utilized in a home environment. A final object of this invention to be specifically enumerated herein is to provide an exerciser which will conform to conventional forms of manufacture, be of simple construction and easy to use so as to provide a device that will be economically feasible, long lasting and relatively trouble free in operation. These together with other objects and advantages which will become subsequently apparent reside in the details of construction and operation as more fully hereinafter described and claimed, reference being had to the accompanying drawings forming a part hereof, wherein like numerals refer to like parts throughout. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a top plan view of a first form of exerciser constructed in accordance with the present invention; FIG. 2 is a longitudinal vertical sectional view taken substantially upon the plane indicated by the section line 2--2 of FIG. 1; FIG. 3 is a side elevational view of a second form of exerciser constructed in accordance with the present invention; FIG. 4 is a longitudinal vertical sectional view of a third form of exerciser constructed in accordance with the present invention; FIG. 5 is a perspective view of the exerciser illustrated in FIG. 4; FIG. 6 is an elevational view of an anchoring structure for anchoring the limb exercising ropes of the exerciser to the weight suspension rope of the exerciser illustrated in FIG. 3; FIG. 7 is a fragmentary perspective view of a modified form of support and anchoring structure for a person utilizing the exerciser of the instant invention; FIG. 8 is a perspective view of a further modified form of anchoring structure for a person utilizing the exerciser; FIG. 9 is a side elevational view of a fourth form of exerciser constructed in accordance with the present invention; FIG. 10 is a front elevational view of the assemblage illustrated in FIG. 9; FIG. 11 is a fragmentary perspective view of a fifth form of exerciser constructed in accordance with the present invention; FIG. 12 is a fragmentary enlarged horizontal sectional view taken substantially upon a plane passing centrally through the doorway mounted anchor bar assembly illustrated in FIG. 11; FIG. 13 is a perspective view illustrating the manner in which a single limb to be exercised may be operatively connected to the weight supporting rope of the exerciser; and FIG. 14 is a perspective view of a bar attachment which may be utilized in conjunction with the several forms of exercisers disclosed in order to enable simultaneous exercising of two limbs in opposition to the weight loading of the exerciser. DETAILED DESCRIPTION OF THE INVENTION With reference now more specifically to FIGS. 1 and 2 of the drawings there will be seen first form of exerciser constructed in accordance with the present invention and generally referred to by the reference numberal 10. The exerciser 10 includes a first horizontal frame structure or section referred to in general by the reference numeral 12 and a second upstanding frame structure or section referred to in general by the reference numeral 14. The section 12 includes a pair of opposite side longitudinal tubular members 16 interconnected by means of transverse tubular members 18, 20 and 22 extending between and secured to the longitudinal member 16 at points spaced longitudinally therealong. The section 14 comprises an inverted U-shaped frame including a pair of depending tubular members 24 interconnected at their upper ends by means of an upper horizontal transverse tubular member or bight portion 26 and the lower ends of the tubular members 24 are secured to the ends of the tubular members 16 remote from the transverse tubular member 18. Further, inclined tubular brace members 30 are connected between the opposite ends of the bight portion 26 and mid-portions of the corresponding longitudinal tubular members 16. A platform 32 bridges and is secured to the ends of the tubular members 16 outwardly from the inclined brace members 30 and the platform 32 has a load cushioning pad 34 disposed thereon and extending longitudinally therealong. The end of the pad remote from the section 14 is anchored to the transverse tubular member 18 by means of an anchoring tension member 36 and the end of the pad 34 adjacent the section 14 is lapped over and secured to the lower outwardly projecting horizontal flange 38 of an L-shaped head stop 40 including an upstanding flange 42 having its inner surface cushioned as at 44. A pulley assembly 46 is suitably anchored to the mid-portion of the bight portion 26 and rotatably journals a pulley wheel 48 over which the mid-portion of a flexible line 50 is trained. One end of the line 50 depends downwardly between the legs 24 and has a predetermined amount of weight in the form of a body 51 supported therefrom. The other end of the line 50 inclines outwardly and downwardly from the bight portion 26 toward the end of the section 12 remote from the section 14. That free end of the line 50 is anchored as at 53 to the short bridle 54 having its opposite ends supported from anchor eyes 56 carried by the opposite ends of an anchor assembly referred to in general by the reference numeral 58 and illustrated in greater detail in FIG. 6. The anchor assembly 58 comprises a bolt 60 including a shank 62 having a head 64 at one end. The anchor eyes 56 are disposed on the opposite ends of the shank 62 between thrust washers 66 and the eye mounting portions 68 of a pair of pulley assemblies referred to in general by the reference numerals 70 are mounted on the shank 62 inwardly of the innermost washers 66. A thrust washer 72 is disposed on the shank 62 inwardly of each eye 68 and a plastic spacing sleeve 74 is disposed on the shank 62 inwardly of each washer 72. Further, a center anchor eye 76 is disposed on the shank 62 between the adjacent ends of the sleeves 74 with washers 78 mounted on the shank 62 between the sleeves 74 and the anchor eye 76. If it is desired, a safety line 80 may have one end thereof anchored to the eye 76 and the other end thereof anchored to the bight portion 26. Each of the pulley assemblies 70 includes a journaled pulley wheel 82 and the mid-portion of a flexible tension member 74 is trained over each pulley wheel 82. The free ends of the tension members 84 are equipped with foot and hand engageable loops 86 whereby the hands and feet of a person 88 disposed on the pad 34 may be anchored relative to the tension members 84. Of course, the exerciser 10 illustrated in FIGS. 1 and 2 may then be used to exercise the arms and legs of the person 88 with movement of each leg being opposed by opposite movement of the corresponding arm and all limbs being exercised or only those limbs engaged with one of the tension members 84 may be preloaded by the weight body 51. The padded flange 44 is engaged by the head of the person 88 and the pad 34 is anchored relative to the transverse tubular member 18 thereby preventing movement of the person 88 toward the section 14. With attention now invited more specifically to FIG. 3 of the drawings, there will be seen a second form of exerciser referred to in general by the reference numeral 10'. The exerciser 10' is similar to the exerciser 10 except that the section 12 is replaced by a floor section 12' of a building and the ceiling structure of section 14' of that building has an anchor hook 15 supported therefrom to which a suspension line 17 is connected, the lower end of the suspension line 17 supporting a pulley assembly 46' therefrom corresponding to the pulley assembly 46. In addition, the pulley assembly 46' is also anchored to a vertical wall 13 extending between the floor 12' and the ceiling 14' by means of a line 19 secured at one end to the pulley 46' and at the other end to an anchor eye 21 engaged in the wall 13. A line 50' corresponding to the line 50 extends over a journaled pulley wheel 48' of the pulley assembly 46' and one end of the line 50' supports a hollow container 51' therefrom which may be variously filled with water 52 or sand, etc. to obtain a desired weight. The end of the line 50' remote from the container 51' is anchored to the bridle 54' of an anchor assembly referred to in general by the reference numeral 58' and corresponding to the anchor assembly 58. Additionally, the exerciser 10' includes a pad 34' and anchor member 36' corresponding to the pad 34 and anchor member 36. However, the anchor member 36' is anchored to the floor structure 12' by means of an anchor eye 37. Further, the pad 34' includes a head stop 40' corresponding to the head stop 40 and including a padded upstanding flange 42'. Thus, it may be seen that the exerciser 10' may be used in substantially the same manner as the exerciser 10. With reference now more specifically to FIGS. 4 and 5 of the drawings there may be seen a third form of exerciser referred to in general by the reference numeral 110. The exerciser 110 differs from the exercisers 10 and 10' in that an upstanding cabinet 111 is provided including a hinged front wall section 112 and a top wall 114. The front wall section 112 is hinged to the forward marginal edge portion of the lower wall 119 of the cabinet 111 as at 121 for movement between the open horizontal position thereof illustrated in FIG. 4 of the drawings to an upstanding position closing the opening 123 formed in the front wall 125 of the cabinet 111. A pulley assembly 146 corresponding to the pulley assembly 46 is supported from the inner surface of the top wall 114 and a line 150 corresponding to the line 50 is trained over the pulley wheel 148 of the pulley assembly 46 and supports a bucket or container 151 corresponding to the container 51 at one end with the other end of the line 150 extending through the opening 123 and being anchored to the bridle 154 of an anchor assembly 158 corresponding to the anchor assembly 58 and having a pair of ropes or tension members 184 operatively engaged therewith corresponding to the members 84 and having hand and foot engageable loops 186 on their free ends corresponding to the loops 86. The cabinet 110 may be anchored relative to a horizontal support surface by means of suitable anchor structure 127 secured through the bottom wall 119 of the cabinet 111. In addition, if the anchor structure 127 is not provided or if additional bracing between the front wall section 112 and the cabinet 111 is desired, inclined braces 129 may be secured between opposite side portions of the front wall 125 of the cabinet 111 and the inner surfaces of the front wall section 112 when the latter is in the open horizontal position illustrated in FIG. 4. Further, a head stop 140 corresponding to the head stop 40 is supported on the inner surface of the free swinging edge portion of the front wall section 112 and has a pad 134 operatively associated therewith corresponding to the pad 34, the pad 134 being rollable into a stored position thereof such as that illustrated in FIG. 5 and the bracing members 128 being removable from their supporting sockets 141 and 143 and receivable within the cabinet 111 before the front wall section 112 is swung to the closed position closing the opening 123, a latch structure 145 being provided on the front wall 125 for retaining the front wall section 112 in a closed position. Further, the line 150 and anchor assembly 158 as well as the ropes 184 may be wholly contained within the cabinet 111 with the container 151 when the cabinet 111 is closed. Of course, the exerciser 110 may be used in substantially the same manner as the exercisers 10 and 10'. With attention now invited more specifically to FIGS. 9 and 10 of the drawings there will be seen a fourth form of exerciser referred to in general by the reference numeral 210. The exerciser 210 is similar to the exerciser 10' except that the head stop 240 thereof is braced relative to the upstanding wall 13 by means of a rod 241, a pad 234 corresponding to the pad 34' being utilized in conjunction with the head stop 240. In addition, the anchor assembly 258 corresponding to the anchor assembly 58' has its safety line 280 anchored to the ceiling 214 by means of an anchor eye 281 and the line 250 passes over the pulleys 248 of a pair of pulley assemblies 246 anchored relative to the ceiling 214 with the two pulley assemblies 246 spaced along the wall 13. Also, the anchor assembly 258 includes lines 284 and loops 286 corresponding to the lines and loops 84 and 86 and a weight 251 corresponding to weight 151. With attention now invited more specifically to FIGS. 11 and 12 of the drawings, it may be seen that a fifth form of exerciser referred to in general by the reference numeral 310 may be provided. The exerciser 310 differs from the three previously described exercisers only in that the line 350 thereof is passed over the pulley wheel 348 of a pulley assembly 346 anchored to the mid-portion of an extendable rod assembly referred to in general by the reference numeral 351 mounted in the upper portion of a doorway opening 353 formed in a wall 355. The weighted end of the line 350 supports a container for weight 357 corresponding to the weight 52 and the rod assembly 351 comprises a first tubular section 360 having an abutment block 362 supported from one end thereof, the abutment block 362 being padded with a sheet 364 of resilient material secured thereto by means of nails 366. The end of the tubular member 360 remote from the abutment block 362 has one end of an externally threaded rod 368 telescoped thereinto, a threaded thrust nut 370 being threaded on the end of the rod 368 projecting outwardly of the tubing member 360 and being provided with a laterally directed handle 372 for applying rotational torque to the nut 370. The end of the rod 368 remote from the end thereof telescoped into the tubular member 360 is embedded in an abutment block 384 corresponding to the block 362 and which is also provided with a resilient covering 386 corresponding to the covering 364. The end of the rod 368 telescoped into the tubing member 360 includes an eye member 388 and the end of the tubing member 360 embedded in the abutment block 362 includes a similar eye member 390. An elastic tension member 392 is connected between the eye members 388 and 390 and thereby urges the rod 368 toward its maximum retracted position within the tubular member 360 as determined by the position of the nut 370 on the rod 368. The rod assembly 351 is disposed within the opening 353 and then expanded into selected position within the upper part of the opening 353 by threading the nut 370 on the rod 368 in order to forcibly extend the rod 368 relative to the tubular member 360. Of course, the center portion of the rod assembly 351 has an anchor eye 396 supported therefrom by which the pulley assembly 346 is supported from the rod assembly 351. The exerciser 310 may include a user supporting structure such as that provided in conjunction with the exerciser 10' and will of course be usable by the user of the exerciser 310 is substantially the same manner as the exercisers 10, 10', 110 and 210 are used. With attention now invited more specifically to FIG. 13, it will be seen that a handgrip ring 400 including a resilient handgrip portion 402 may be directly connected to a line 450 corresponding to one of the lines 50, 50', 150, 250 or 350 in the event only a single limb of the person using the exerciser is to be exercised at any given time. In addition, an elongated rod 404, see FIG. 14, having opposite end handgrips 406 and a center anchor eye 408 may be directly connected to the line 450, if desired. In this manner, the arms of the user of the exerciser may simultaneously exercise both arms in the same manner. With attention now invited more specifically to FIG. 7 of the drawings, it may be seen that the head stop 40 may have a pair of shoulder abutment boards 40" equipped with pads 42" on one pair of corresponding ends and L-shaped mounting brackets 44" on the other pair of corresponding ends thereof removably engaged with the upstanding flange 42 of the head stop 40. Of course, the upper portions of the shoulders of the user 88 of FIG. 2 may be engaged with the pads 42" of FIG. 7 while the head of the user 88 is engaged with the pad 44 of the head stop 40. With attention now invited more specifically to FIG. 8 of the drawings there may be seen a waist-encircling belt 90 which may be removably secured about the waist of the user of any of the exercising devices hereinbefore described. One end of an anchor tension member 92 is secured to the belt 90 and the other end of the anchor tension member 92 may be utilized in the same manner as the members 36 and 36'. The various lightweight containers 51, 151 and 251 may be filled with water or said, etc. to achieve the desired weight value by the user. Further, the amount of exercise which may be performed in a given time may be varied by varying the speed at which exercises are carried out. Also, a pair of limbs being exercised are opposed by the specific weight used and the exerciser should be used in a manner to exercise one or two pairs of limbs while the specific weight being used is maintained at least substantially motionless, thereby also developing coordination. 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 horizontal first structure and a second structure elevated above the first structure are provided and the elevated second structure supports a pulley assembly therefrom including a journaled pulley. Abutment structure is provided on the first horizontal spaced normal from an upstanding plane containing the pulley assembly and against which a person disposed on the first horizontal structure may abut at least a portion of his body to prevent sliding of that person relative to the first horizontal structure toward the aforementioned plane. An elongated flexible tension member has its mid-portion passed over the journaled pulley and a first end portion of the tension member depends downwardly from the journaled pulley and supports a weight body therefrom. The second end portion of the tension member extends downwardly away from the journaled pulley in a direction inclined away from the aforementioned plane and the lower end of the second end portion of the tension member includes structure adapted to be engaged by the free end portion of at least one limb of a person disposed on the first horizontal structure. | 5,294 | 227 | big_patent | en |
Summarize: By. Daily Mail Reporter. PUBLISHED:. 15:33 EST, 15 August 2013. |. UPDATED:. 16:04 EST, 15 August 2013. The lawyer for one of the two 18-year-olds charged with circulating rape pictures of a teenage girl who later hanged herself says his client is being persecuted by a 'cyber-lynch mob.' The tragic suicide in April of Rehtaeh Parsons, a 17-year-old high school student from Nova Scotia, Canada, brought international attention to her claims that she was gang-raped when she was 15 and then relentlessly tormented by classmates who called her a'slut' and harassed her for sex. Much of the bullying was a result of a photo the circulated at her school that showed Parsons being assaulted by a teen boy while throwing up out a window. The two 18-year-olds are charged with child pornography. One is accused of taking the picture, both are accused of circulating it. Because the alleged crime took place when the men were 16, they are charged as juveniles and their identities cannot be revealed. 'Victim': She was labelled a'slut' after she was allegedly gang raped by four teenagers in 2010. Protection: The two teenage suspects, now 18, cannot be identified under Canadian law because they were 16 when the alleged crime took place. They were both in court today with their parents for a brief appearance. The judge set a trial for September 19. Josh Arnold, the attorney for one of the men, told CTV News that his client has already been convicted in the court of public opinion. 'The Internet mavens and some members of the media have acted like a lynch mob in relation to this matter. They've tried and convicted this person without any due process whatsoever,' he said. Rehtaeh's heartbroken parents say that she hanged herself in their bathroom two years after the alleged attack because she could not bear to bullying and torment at school. Her uncle was furious about the attorney's allegations. 'This man's saying that these boys have been prosecuted in the media.That hasn't been the case. That's not true,' Michael Parsons said. A handful of protestors turned out to the courthouse where the suspects in the Rehtaeh case were due to appear. 'This case has been brought forward in the media. Because, it was only through the media participation that this case is even being looked at today.' He also said he is praying for the two accused. 'I have to pray for these boys. I have to pray for them to become good men. That's the hardest thing for me to do. But it's something I have to do. That's what's hard. If you want to know the truth that's the hardest thing to do,' he told the CBC. 'I have absolutely no faith in the RCMP or the Halifax city police or the Crown prosecutors because they totally dropped this case, they totally dropped the ball.' Retaeh's death led to an outcry because no one was charged or even reprimanded at the school - either for her rape or for the bullying. Tragedy: Rehtaeh Parsons, 17, took her life in April after enduring months of bullying after an alleged gang rape. Four boys, aged 14, 17 and two aged. 16, allegedly took it in turns to sexually assault her on November 12, 2011, after plying her with. alcohol, she told her mother days after. Her. mother said she saw an image of the alleged attack which showed her. daughter being raped by a teenage boy as he gave a 'thumbs up' sign while she vomited out of a window. Police initially concluded there were no grounds to charge anyone after a year-long investigation. Royal Canadian Mounted Police Cpl Scott MacRae said they arrested two. males today. Investigators said they are being questioned and no. further information will be released at this time. 'Due to the sensitive nature around this investigation, the. investigators do want to ensure that no court process is affected,' MacRae said. The arrests came after someone came. forward in April and provided new information and said they were willing. to verify who the suspects are. 'We're just hopeful there's charges laid and others to arrest, hoping. that they're finally willing to tell their side of the story,' Leah. Parsons, Rehtaeh's mother, said. Innocence: Rehtaeh is pictured as a baby with her parents, Leah Parsons and Clen Canning. Distraught: Rehtaeh's mother chokes back tears at the teenager's funeral in April after she hanged herself. 'A sense of relief came over me that at least they're going to be questioned.' Last month Leah and her husband spoke to People magazine about their daughter's tragic death. 'When Rehtaeh was born, I promised her the world. Now my beautiful girl is gone,' Leah said. She started smoking pot to 'numb her emotions' and deal with Facebook messages and texts that came through by the dozen. They read things like: 'Sluts are not welcome here' and 'everyone knew exactly what you have done'. Rehtaeh. eventually closed down her Facebook page because there were so many men. asking her to have sex with them. They would say: ‘You had sex with my. friend, so why not me?’ The response from the school was, apparently, to do very little. Halifax school board spokesman Doug Hadley has said that none of the boys were spoken to. Her father, Glen Canning, told People: 'My. daughter wasn't bullied to death, she was disappointed to death. Disappointed in people she thought she could trust, her school, and the. police.' Left behind: Her father said she killed herself out of disappointment in her. school, her former friends and law enforcement officers who failed to. help her. Rehtaeh's death prompted the Nova Scotia government to launch reviews of. the RCMP's original investigation and the school board's handling of. the matter. An. independent review released in June concluded the Halifax Regional. School Board could have done a better job, but it was hindered by the. fact that Rehtaeh was often absent from class. 'No help': Cole Harbour High School staff failed to speak to the boys involved and Rehtaeh left | Summary: Rehtaeh Parsons, 17, hanged herself in April. Told her mother she was gang-raped two years before then taunted about it after classmates saw a picture from the assault. Two men were arrested earlier this month and charged with taking and circulating the picture her being raped. Lawyer for one of the men says his client has already been convicted in the court of public opinion. Rehtaeh's uncle says he is praying for the two suspects. | 1,522 | 107 | cnn_dailymail | en |
Summarize: Photo SYDNEY, Australia — Scientists surveying the Great Barrier Reef said Tuesday that it had suffered the worst coral die-off ever recorded after being bathed this year in warm waters that bleached and then weakened the coral. About two-thirds of the shallow-water coral on the reef’s previously pristine, 430-mile northern stretch is dead, the scientists said. Only a cyclone that reduced water temperatures by up to three degrees Celsius in the south saved the lower reaches of the 1,400-mile reef from damage, they added. On some atolls in the north, all the coral has died, said Prof. Terry Hughes, the director of the ARC Center of Excellence for Coral Reef Studies at James Cook University in Townsville, in the eastern state of Queensland. Professor Hughes and a team of scientists drew their findings from about 900 dive surveys along the length of the reef in October and November. “The good news is that in the south, only about 1 percent of the reef’s coral has died, and the mortality rate in the middle is about 6 percent,” Professor Hughes said. Vibrant color has returned to that coral, and the reef there is in good condition, he added. “But in the north, mortality rates are very high, and in some places where coral has survived but it has weakened, the per capita predation rate has gone through the roof,” he said. Masses of Drupella snails could be seen swarming around and eating the remaining healthy coral, he said. Advertisement Continue reading the main story The bleaching was the third such event known to strike the reef, which extends along almost the entire eastern coast of Queensland and is listed as a natural World Heritage Site by the United Nations. Steven Miles, Queensland’s environment minister, called the bleaching a tragedy. “In the north we have a very high coral death rate,” Mr. Miles said at a news conference in Brisbane, the state capital. “The tragedy of this is that the northern sections were the sections least impacted by human impacts, and to see those sections, two-thirds of the northern section, dead is catastrophic.” However, he said, the reef “is a very big system, and the mortality rate varies substantially.” Mr. Miles and the federal environment minister, Josh Frydenberg, who also oversees energy policy in Australia, said Friday that 45 million Australian dollars, about $33.6 million, would be provided to improve water quality and reduce sediment runoff. That announcement precedes a government report, due to be submitted to the United Nations by Thursday, on the health of the reef and the government’s management of threats to it. In May 2015 the United Nations stopped short of putting the reef on an “in danger” list, but it warned that climate change, water pollution and the effects of coastal development were all detrimental. “The government has a staunch commitment to conserving this amazing natural asset,” Mr. Frydenberg said in a written statement on Friday. Photo Some scientists and environmental advocates have criticized the government’s efforts to protect the reef, saying they have fallen far short. They have also pointed to a seeming contradiction in the wishes of the Queensland government to protect the reef even as it pushes ahead with plans to develop the Carmichael coal mine, the country’s biggest, which lies less than 200 miles inland in the Galilee Basin. “Spending $45 million to improve water quality on the reef is like putting a Band-Aid on a person who has cancer,” said William Steffen, a climate scientist at the Australian National University College of Medicine, Biology and Environment. As custodian of the reef, the government has an obligation to manage one of the world’s greatest natural wonders, Dr. Steffen said. “It is nonsense to think we can open up a new coal mine and think we are going to save coral reefs.” Advertisement Continue reading the main story This month, Australia ratified the Paris climate agreement to limit pollution to stop the Earth’s warming by more than 1.5 to 2 degrees Celsius above preindustrial levels, or 2.7 to 3.6 degrees Fahrenheit. Environmentalists have unsuccessfully tried to block the development of the enormous mine. Other countries have pledged to reduce the use of fossil fuels, which contribute to global warming. Burning coal is a major cause of greenhouse gas emissions. Coral in the north was “cooked” as water temperatures rose about two degrees, Professor Hughes of James Cook University said. “That coral did not bleach and die slowly.” Coral in slightly cooler waters bleached more slowly, expelling the tiny algae that give it its color. If the water then cooled quickly enough, the algae returned to recolonize the coral, which recovered. Cyclone Winston, which passed over Fiji in late February before dissipating as it hit the coast of Queensland, brought a change in water temperatures that helped preserve the coral at the south end of the reef. Mr. Frydenberg said Friday that about 22 percent of the entire reef’s coral had died after the bleaching event. But he added that coral cover had increased around 19 percent in the years leading up to it, a figure that Professor Hughes disputes. “The Great Barrier Reef is very resilient and quite strong,” Mr. Frydenberg said in an email on Tuesday. “The Australian and Queensland governments have a Reef 2050 plan, which will see $2 billion invested over the next decade in order to improve the health of the reef.” Greg Torda, a coral researcher at James Cook University, said the biodiversity of the reef had been severely compromised in some regions because flat, or tabletop, and branching corals had died. These corals provide structure for small fish to hide from predators. “Bigger boulder corals, some hundreds of years old, provide light and shade for fish in reef habitats,” Dr. Torda said. “Reefs are structurally very complex, and all shapes on the reef provide important diversity.” Reefs that are damaged tend to flatten out, losing habitat and then species diversity over decades. “We will have a reef in 30 years’ time, but the species along the reef are already shifting,” Professor Hughes said. “And we are already seeing less diversity.” Higher sea temperatures have led to the worst bleaching event on record, new study finds, with coral predicted to take up to 15 years to recover A new study has found that higher water temperatures have ravaged the Great Barrier Reef, causing the worst coral bleaching recorded by scientists. In the worst-affected area, 67% of a 700km swath in the north of the reef lost its shallow-water corals over the past eight to nine months, the Australian Research Council Centre of Excellence for Coral Reef Studies based at James Cook University study found. “Most of the losses in 2016 have occurred in the northern, most-pristine part of the Great Barrier Reef,” Prof Terry Hughes said. “This region escaped with minor damage in two earlier bleaching events in 1998 and 2002, but this time around it has been badly affected.” The southern two-thirds of the reef escaped with minor damage, Hughes said. This part was protected from the rising sea temperatures because of cooler water from the Coral Sea. Scientists expect that the northern region will take at least 10 to 15 years to regain the lost corals but are concerned a fourth bleaching event could interrupt the slow recovery. The dire assessment of the reef’s health comes as the Australian government is due to report to Unesco’s world heritage committee on its handling of the reef. After the federal government submits the report Unesco will decide whether to again consider listing the Great Barrier Reef on its “list of world heritage in danger”. The government will need to report on how it has funded and implemented its Reef 2050 long-term sustainability plan, as well as how the bleaching event has affected the reef. Since it last considered including the Great Barrier Reef on its list, the reef has undergone the worst bleaching event in recorded history. According to government agencies, 22% of the reef was killed in one hit, as unusually warm waters bleached and killed the coral. Climate change at the Great Barrier Reef is intergenerational theft. That's why my son's in this story Naomi Klein Read more Climate change poses such a threat to the reef that the former head of the Great Barrier Reef Marine Park Authority has called for a ban on all new coalmines in Australia to protect the reef from climate change. Graeme Kelleher, who was the first chief executive of the authority, a position he held for 16 years, said: “Australia cannot have a healthy Great Barrier Reef and a continuing coal industry. “I love the reef and I have worked to preserve it since 1979; I will oppose anything that threatens to destroy it,” he said. In this Friday Nov. 25, 2016, photo Australian senator Pauline Hanson listens to marine scientist Alison Jones, left, as she displays a piece of coral on the Great Barrier Reef off Great Keppel Island,... (Associated Press) CANBERRA, Australia (AP) — Warming oceans this year have caused the largest die-off of corals ever recorded on Australia's Great Barrier Reef, scientists said Tuesday. The worst-affected area is a 700-kilometer (400-mile) swath in the north of the World Heritage-listed 2,300-kilometer (1,400-mile) chain of reefs off Australia's northeast coast, said the Australian Research Council Centre of Excellence for Coral Reef Studies. The center, based at James Cook University in Queensland state, found during dive surveys in October and November that the swath north of Port Douglas had lost an average of 67 percent of its shallow-water corals in the past nine months. Farther south, over the vast central and southern regions that cover most of the reef, scientists found a much lower death toll. The central region lost 6 percent of bleached coral and the southern region only 1 percent. "The mortality we've measured along the length of the Great Barrier Reef is incredibly patchy," the center's director, Terry Hughes, told reporters. "There's very severe damage in the northern section of the reef." "The good news is that south of Port Douglas, including the major tourist areas around Cairns and the Whitsundays (Whitsunday Islands), have had relatively low levels of mortality," he added. The governments of Australia and Queensland will update the UNESCO World Heritage Center this week on progress being made to protect and improve the reef, including their response to coral bleaching. Providing a status update to the World Heritage Committee was required as part of its decision in June last year not to list the reef as "in danger." Federal Minister for the Environment and Energy Josh Frydenberg said Tuesday that the reef's coral cover had increased by 19 percent in recent years before it suffered a "significant bleaching event" this year, caused by the El Nino weather effect and climate change. "What that shows is that the Great Barrier Reef is very resilient and quite strong," Frydenberg's office said in a statement. The governments plan to spend 2 billion Australian dollars ($1.5 billion) over the next decade on improving the reef's health. Hughes said the coral death rates in the north would likely make the task of keeping the reef off the "in danger" list much harder. "In its ongoing dialogue with UNESCO, Australia has said the outstanding universal values of reef are in tact because of the pristine condition of the northern reef. That's simply no longer the case," Hughes said. Researcher Andrew Baird said the 2016 coral die-off was "substantially worse" than the previous worst-ever event in 1998. "The proportion of reefs that were severely affected was much, much higher," Baird said, adding that he did not have precise figures immediately available. The 1998 event was restricted to in-shore reefs around the Queensland coastal city of Townsville, while the 2016 destruction affected a much larger area, he said. Scientists expect that the northern region will take at least 10 to 15 years to regain the lost corals. They are concerned that another bleaching event could interrupt that recovery. There have been three extreme mass bleaching events in 18 years on the reef. In each case, the areas that suffered the worst bleaching were where the water was hottest for the longest period of time. Reef tourism operator Craig Stephen did not expect the dead coral would diminish visitors' experience of one of Australia's biggest tourist drawcards. "The patchiness of the bleaching means that we can still provide our customers with a world-class coral reef experience by taking them to reefs that are still in top condition," Stephen said in a statement. Graeme Kelleher, who headed the Great Barrier Reef Marine Park Authority for 16 years, said last week that Australians must not buy the "political lie" that they can have the reef as well as major coal mines nearby. "We've lost 50 percent of the coral cover on the Great Barrier Reef in the last 30 years and the main cause of that is the burning of fossil fuel. I sincerely hope UNESCO rejects the claim that the government is doing enough," Kelleher said. | Summary: This year saw the worst mass die-off of coral ever recorded at the Great Barrier Reef, with the "most-pristine" parts of the reef being hit the hardest, the Guardian reports. One 430-mile section in the northern portion of the 1,400-mile-long reef lost 67% of its shallow-water coral. All told, the Great Barrier Reef lost 22% of its coral in 2016's bleaching event. "To see those sections, two-thirds of the northern section, dead, is catastrophic," an Australian environmental minister tells the New York Times. The northern section of the reef had historically been the least damaged by human activity. The coral bleaching was caused by warming waters; the southern portion of the reef escaped great harm thanks to a cyclone that lowered water temperatures. This was the third-and by far the worst-coral bleaching event to hit the Great Barrier Reef in the past 18 years, the AP reports. Since the last event in 2002, the reef's coral had increased by 19%, according to the Australian government. Experts say it could take 10 to 15 years to regain the coral lost in this year's bleaching event-but that's assuming there isn't a fourth during that span. One expert says that even if the Great Barrier Reef hits pre-bleaching levels of coral, it will never regain the diversity it once had. The UN is considering adding the Great Barrier Reef to its list of world heritage sites that are in danger. And the Australian government plans to spent $1.5 billion on protecting the reef over the next decade. | 3,013 | 382 | multi_news | en |
Summarize: « Previous Main Next » Mike Huckabee Blasts Student Newspaper for ‘Sensationalizing’ Comments on Gay Marriage ABC News’ Huma Khan reports: In an unusual battlefront, former Arkansas governor Mike Huckabee is embroiled in a heated war of words with a college newspaper after telling a student reporter that it is “disingenuous” to recognize gay marriage but not polygamy. Huckabee told The Perspective, the College of New Jersey’s student magazine, that it’s not his place to tell someone how to live, but he argued against the legitimacy of civil unions. “There’s a real level of being disingenuous on the part of the gay and lesbian community,” he said. "That would be like saying, well there's there are a lot of people who like to use drugs so let's go ahead and accommodate those who want to use drugs. There are some people who believe in incest, so we should accommodate them. There are people who believe in polygamy, should we accommodate them?... Who gives you the right to say that a Polygamist is not just as right in his argument as is the person who wants same sex [marriage]?” The former GOP presidential candidate also spoke out strongly against gay adoption. “This is not about trying to create statements for people who want to change again the basic fundamental definitions of family,” he said. “Children are not puppies. This is not a time to see if we can experiment and find out how does this work?” Huckabee isn’t new to such controversy. When running for the Senate in 1992, he wrote in a questionnaire for the Associated Press that homosexuality is “aberrant, unnatural, and sinful lifestyle.” He said that AIDS research was receiving too much money and that celebrities should pay for it rather than the federal government. As governor, Huckabee supported a policy that kept same-sex couples from becoming foster parents. Huckabee hasn’t been shy about his views on gay marriage, but he said the college newspaper “grossly distorted” them when it published the interview on April 9. "The young college student hopefully will find a career other than journalism,” Huckabee wrote on his web site. “Not only did he attempt to sensationalize my well known and hardly unusual views of same-sex marriage, he also inaccurately reported my views on Michael Steele as GOP chairman - I offered my support and didn't ‘Rip into Steele’ as his article asserted.” On whether the embattled RNC chairman should resign, Huckabee told the student reporter it’s up to RNC members, but that “It’s indefensible the way some of the money has been spent.” Michael Tracy, editor-in-chief of The Perspective, defended the publication and blasted Huckabee for resorting to “ad hominem attacks.” “Politicians in damage-control mode often stoop to attacking the media so they might avoid being accountable for the substance of their remarks,” Tracy wrote. Do you think Huckabee’s statements were blown out of context? Listen to the audio of parts of the interview posted by The Perspective: Huckabee Rips Steele, Romney, LGBT Activists Calls Romney’s Healthcare Plan “Dismal Failure,” Compares Same-Sex Marriage to Incest By M.C. TRACEY UPDATE: PERSPECTIVE RESPONDS TO HUCKABEE CLAIM THAT HIS VIEWS WERE ‘GROSSLY DISTORTED’ IN ARTICLE (4.13.10) In an interview Wednesday, former Arkansas governor and Republican presidential candidate Mike Huckabee weighed in on embattled Republican National Committee Chairman Michael Steele, slammed his potential 2012 presidential primary rival Mitt Romney, and reiterated strong opposition to same-sex marriage and the repeal of ‘Don’t Ask Don’t Tell.’ The popular Fox News host, who appeared at The College of New Jersey in Ewing, NJ, leveled into Steele for his handling of last week’s revelation that RNC officials spent donor money at a bondage-themed Los Angeles strip club. “I think what’s happened is horrible,” Huckabee said. “The question is, how is [Steele] going to take control and how is he going to explain it? I think there does need to be a better explanation than what’s come forth so far,” he continued. “It’s been pretty weak.” Huckabee said ongoing controversies involving Steele may become an electoral liability for Republicans as this year’s midterm elections approach. “It’s indefensible the way that some of the money has been spent,” Huckabee said, though he declined to call for Steele’s resignation. “Whether he should resign really is a question for the RNC, and I’m not a member, so I’m not going to get into that.” Huckabee also blasted fellow presidential aspirant Mitt Romney, who as governor of Massachusetts signed into law a healthcare plan that closely resembles Congress’ recently passed reforms. “Essentially ‘Obamacare’ and what Massachusetts has is virtually identical,” Huckabee said, characterizing the system Romney supported as a “dismal failure.” “It’s resulted in huge cost increases, exponentially beyond what was projected,” he charged. Romney’s inconsistent public comments on the matter, Huckabee continued, is likely to be a concern for Republican primary voters in 2012. “On one hand, [Romney] said the Massachusetts plan is not like the Obama plan. Then I heard him say, ‘Well, it’s similar, but we’ve learned a lot from it since then.’ So he’s going to have to come up with one answer that deals with it,” Huckabee said. “I think he’s got to make the explanation as to why he believed it was a great idea.” Huckabee, a former Baptist preacher, remains in favor of keeping in place ‘Don’t Ask Don’t Tell,’ the policy that requires the military to expel openly gay service members. “I wouldn’t support a repeal if I were commander-in-chief,” he said. “You don’t see foot soldiers out there demanding it. I’m not sure that’s the most important thing we ought to be doing for the military.” “[‘Don’t Ask Don’t Tell’] touches an extraordinarily small group of people,” Huckabee continued. He dismissed calls to amend the policy as “primarily a posturing point for political purposes,” and an attempt to “force something on the military that they themselves haven’t pushed that hard.” “I think we certainly should be very sensitive to the fact that the purpose of the military is not to see if we can create social experiments,” Huckabee warned. He continues to oppose any government recognition of same-sex relationships. Even civil unions are “not necessary,” Huckabee said. “I think there’s been a real level of being disingenuous on the part of the gay and lesbian community with their goal of civil unions,” he alleged, referring to LGBT activists who first claimed that their goal in several states was to enact civil unions, but subsequently launched efforts to implement full marriage rights. Huckabee went on to draw parallels between homosexuality and other lifestyles that are considered by some to be morally aberrant. “You don’t go ahead and accommodate every behavioral pattern that is against the ideal,” he said of same-sex marriage. “That would be like saying, well, there are a lot of people who like to use drugs, so let’s go ahead and accommodate those who want to use drugs. There are some people who believe in incest, so we should accommodate them. There are people who believe in polygamy, so we should accommodate them.” He also affirmed support for a law in Arkansas that prohibits same-sex couples from becoming adoptive or foster parents. “I think this is not about trying to create statements for people who want to change the basic fundamental definitions of family,” Huckabee said. “And always we should act in the best interest of the children, not in the seeming interest of the adults.” “Children are not puppies,” he continued. “This is not a time to see if we can experiment and find out, how does this work?” In what may come as a surprise for some, Huckabee agreed that an atheist could be fit to serve as president. “I’d rather have an honest atheist than a dishonest religious person,” he said. “It’s better to have a person who says, ‘Look, I just don’t believe, and that’s where my honest position happens to be,’” he said. “I’m frankly more OK with that than a person who says, ‘Oh, I am very much a Christian. I very much love God.’ And then they live as if they are atheists, as if they have no moral groundings at all. That’s more troubling.” “I think it’s nice if a person believes in God,” Huckabee said. “I’d hate to think somebody was making decisions who thought that he couldn’t be higher than himself.” | Summary: It's no Jersey Shore, but Mike Huckabee is facing down a bunch of New Jersey college kids whose student newspaper he says "sensationalized" his thoughts on gay marriage. The former governor, in an interview with the College of New Jersey's Perspective, said that not every group's needs should be accommodated, drawing parallels between gay marriage and drug use, polygamy, and incest, as well as calling gay activists "disingenuous." "Who gives you the right to say that a polygamist is not just as right in his argument as is the person who wants same sex" marriage? Huckabee asked. He later took issue with the way his remarks had been reported and fired off a statement claiming his views had been "grossly distorted," and that the student he spoke to "hopefully will find a career other than journalism," ABC reports. | 2,052 | 191 | multi_news | en |
Write a title and summarize: The interaction among multiple microbial strains affects the spread of infectious diseases and the efficacy of interventions. Genomic tools have made it increasingly easy to observe pathogenic strains diversity, but the best interpretation of such diversity has remained difficult because of relationships with host and environmental factors. Here, we focus on host-to-host contact behavior and study how it changes populations of pathogens in a minimal model of multi-strain interaction. We simulated a population of identical strains competing by mutual exclusion and spreading on a dynamical network of hosts according to a stochastic susceptible-infectious-susceptible model. We computed ecological indicators of diversity and dominance in strain populations for a collection of networks illustrating various properties found in real-world examples. Heterogeneities in the number of contacts among hosts were found to reduce diversity and increase dominance by making the repartition of strains among infected hosts more uneven, while strong community structure among hosts increased strain diversity. We found that the introduction of strains associated with hosts entering and leaving the system led to the highest pathogenic richness at intermediate turnover levels. These results were finally illustrated using the spread of Staphylococcus aureus in a long-term health-care facility where close proximity interactions and strain carriage were collected simultaneously. We found that network structural and temporal properties could account for a large part of the variability observed in strain diversity. These results show how stochasticity and network structure affect the population ecology of pathogens and warn against interpreting observations as unambiguous evidence of epidemiological differences between strains. Interactions between strains of the same pathogen play a central role in how they spread in host populations. [1–7]. In Streptococcus pneumoniae and Staphylococcus aureus, for instance, several dozen strains can be characterized for which differences in transmissibility, virulence and duration of colonization have been reported in some cases [8,9]. Strain diversity may also affect the efficacy of prophylactic control measures such as vaccination or treatment. Indeed, strains may be associated with different antibiotic resistance profiles [3,5, 10,11], and developed vaccines may only target a subset of strains [2,3, 12]. With the increasing availability of genotypic information, it has become easy to describe the ecology of population of pathogens and to monitor patterns of extinction and dominance of pathogen variants [13–17]. However, the reasons for multi-strain coexistence patterns (e. g. coexistence between resistant and sensitive strains) or dominance of certain strains (e. g. in response to the selection pressure induced by treatment and preventive measures) remain elusive. One may invoke selection due to different pathogen characteristics, but also environmental and host population characteristics, leading to differences in host behavior, settings and spatial structure may affect the ecology of strains [14–19]. In particular, human-to-human contacts play a central role in infectious disease transmission [20]. This is increasingly well described thanks to extensive high-resolution data—including mobility patterns [21–23], sexual encounters [24], close proximity interactions in schools [25,26], workplaces [27], hospitals [16,28–31], etc. —that enable basing epidemiological assessment on contact data with real-life complexity [32,33]. For instance, the frequency of contacts can be highly heterogeneous leading more active individuals to be at once more vulnerable to infections and acting as super-spreaders after infection [24,33–35]. Organizational structure of certain settings (school classes, hospital wards, etc.) and other spatial proximity constraints lead to the formation of communities that can delay epidemic spread [36,37]. Individual turnover in the host population is also described as a key factor in controlling an epidemic [20,38]. It is likely that, since they impact the spread of single pathogens, the same characteristics could affect the dynamics in multi-strain populations. It was shown, indeed, that network structure impacts transmission with two interacting strains [39–46], the evolution of epidemiological traits [47–49] and the effect of cross-immunity [50,51]. Yet in these cases, complex biological mechanisms—such as mutation, variations in transmissibility and infectious period, cross immunity—were used to differentiate between pathogens, thereby making the role of network characteristics difficult to assess in its own right. For this reason, we focused on the dynamical pattern of human contacts and examined whether it contributes to shaping the population ecology of interacting strains under minimal epidemiological assumptions regarding transmission. We described a neutral situation where all strains have the same epidemiological traits and compete via mutual exclusion (concurrent infection with multiple strains is assumed to be impossible) in a Susceptible-Infected-Susceptible (SIS) framework. We studied the spread of pathogens in a host population during a limited time window, disregarding long-term evolution dynamics of pathogens. More precisely, new strains were introduced through host turnover rather than de novo mutation or recombination in pathogens. We quantified the effect of network properties on the ecological diversity in strain populations with richness and dominance indicators. We assessed in turn heterogeneities in contact frequency, community structure and host turnover by comparing simulation results obtained with network models exhibiting a specific feature. We then interpreted S. aureus carriage in patients of a long-term care facility in the light of these results. We simulated the stochastic spread of multiple strains on a dynamical contact network of individuals (nodes of the network). Individuals can be either susceptible or infected with a single strain at a given time, and, for each strain, β and μ indicate the transmission and the recovery rate respectively. We assumed turnover of individuals, who enter the system with rate λin, and associated injection of previously unseen strains, carried by incoming individuals with probability ps. We considered synthetic network models, each displaying a specific structural feature, as well as a real network reconstructed from close-proximity-interaction data collected in a hospital facility. We calibrated all network models to the same average quantities—average population size V ¯, fraction of active nodes a ¯, average degree k ¯ and strength of the community repartition pIN, when applicable—that were chosen to correspond with the hospital network used in the application. Epidemiological parameters were motivated by the duration of S. aureus carriage in patients. A larger range of values was explored in some cases to address their impact on the dynamics. We analyze the structure of strain population at the dynamic equilibrium by computing, for each network model, ecological diversity measures, including species richness and evenness/dominance indices [52,53]. All details about network models, numerical simulations and ecological indicators are described in the Materials and methods section. In order to probe the effect of contact heterogeneity on strain ecology we compared a homogeneous model (HOM) in which all nodes have the same activity potential, i. e. they have equal rate of activation to establish contacts, with a heterogeneous model (HET), akin to the activity-driven model described in [34], where the activity potential is different across nodes and is drawn from a power-law distribution. Fig 1 shows the results of numerical simulations comparing HOM and HET models. Sample epidemic trajectories are reported in Fig 1A. Here every strain is indicated with its own color to display its dynamics resulting from the interaction with the other strains. Fig 1B–1D shows summary statistics in varying strain transmissibility β. The prevalence presents a well-known behavior for both static and dynamic networks (Fig 1B): contact heterogeneities lower the transmissibility threshold above which total prevalence is significantly above zero, thus allowing the spread of pathogens with low transmissibility. At the same time, however, heterogeneities hamper the epidemic spread when β is large, reducing the equilibrium prevalence [35]. Fig 1C shows the average richness, i. e. the number of distinct strains co-circulating. For low values of β HET displays larger richness values compared to HOM. This trend reverses as β increases, and the richness is lower in HET consistently with the lower level of prevalence. The relation between richness and prevalence, however, is not straightforward. For instance, the reduction in richness for high β values is important even for the case with limited contact heterogeneity, when prevalence is barely affected. The scaling between prevalence and richness is not linear as β varies (Fig 1D), and the relation between the two quantities varies appreciably among contact networks. In correspondence of a fixed value of prevalence, heterogeneous networks have lower richness—e. g. a prevalence value of ∼0. 8 corresponds to ∼20% lower richness in HET with respect to HOM, as highlighted in Fig 1D. This fact can be explained by the dynamical properties of epidemics on heterogeneous networks. Active nodes, involved in a larger number of contacts, get infected more frequently [35]. Also, a randomly chosen node is likely surrounded by active nodes [33]. As a consequence, injected strains often find their propagation blocked by active infected nodes. In this way, contact heterogeneities enhance the competition induced by mutual exclusion and hamper the wide-spread of emerging strains, similarly to what was found in [46]. This mechanism is further confirmed by looking at the persistence time of strains (S2 Fig in the supporting information). Above the epidemic threshold, it is on average shorter in heterogeneous networks than in homogeneous ones. The distributions are however more skewed in heterogeneous networks, indicating that more strains are going extinct rapidly, while a few others can survive for a long time in the population. If on the one hand hubs accelerate the extinction of certain strains, on the other they act as super-spreaders, amplifying the propagation of other strains. We find that this impacts profoundly the distribution of strains’ abundances, i. e. the strain-specific prevalence. Fig 2A shows that the latter is broader for the HET network, with the most abundant strain reaching a larger proportion of cases. This situation is synthesized by the Berger-Parker index, that quantifies the level of unevenness or dominance of a given ecological system [52,53]. This is defined as the relative abundance of the most abundant strain (see Materials and methods section). Fig 2B shows that Berger-Parker index increases with β for all networks. This is expected since at low β strains′ transmission chains are short and barely interact, while they interfere more at higher values of transmission potential. The Berger-Parker index is always higher in a heterogeneous network, even when the comparison is made at fixed values of richness (Fig 2C). An alternative indicator, the Shannon evenness, shows a similar behavior as displayed in S3 Fig. The fraction of strains going extinct also depends on stochastic effects in a finite size population. We indeed found that increasing network size, when temporal and topological properties were the same, led to an increase in both persistence time and richness (S4 Fig). This shows that interference among transmission chains is reduced in larger populations. However, the relative abundance distribution remained similar, showing that it is primarily affected by the nodes’ activity distribution (S5 Fig). Eventually, we tested whether additional mechanisms of strain injection were leading to different results. In S6 Fig we assumed new strains to infect susceptible nodes already present in the system with rate qs, mimicking in this way transmissions originating from an external source, as it can happen in real cases. The plot of S6 Fig shows the same qualitative behavior described here. We considered a community model (COM) with nC communities in which all nodes are as active as in HOM, but direct a fraction pIN of their links within their community and the rest to nodes in the remaining nC − 1 communities. The closer pIN is to 1, the stronger the repartition in communities is. Fig 3A and 3B shows that a network with communities displays a higher richness for large β; even when community structure barely affects prevalence (Fig 3B). However, the effect is important only when communities are fairly isolated (pIN = 0. 99) and the injection from the outside is not so frequent—otherwise the effect is masked by strain injection which occurs uniformly across communities. In particular, for the values of pIN = 0. 78 and ps = 0. 079, chosen to match the hospital application, the difference with the homogeneous case is very small. The limited role of community structure is also confirmed by the fact that once this feature is combined with heterogeneous activation—in a model with the activation scheme of HET and the stub-matching of COM—the latter property has the dominant effect and the richness decreases (S1 Fig). The relation between richness and prevalence remains the same when adding the injection of new strains due to the transmission from an external source. This mechanism further increases the richness. When β is high and the fraction of infected nodes is close to one, however, such a mechanism is hindered by the fact that susceptible nodes, that can get infected from the external source, are rare (see S6 Fig). This is why richness starts to decrease for high values of β. We tested the consequences of communities in strain dominance by plotting the Berger-Parker index in Fig 3C. For low β, the behavior of the Berger-Parker index follows the trend in richness. The initial decrease in this indicator is due to the increase in richness, that occurs at constant prevalence and is thus associated to a decrease in the average abundance [54]—green curve corresponding to pIN = 0. 99 and ps = 0. 01. At larger values of β, instead, increased competition levels induced higher dominance levels. The increase in strain diversity is due to the reduced competition among strains introduced in different communities. When coupling among communities is low, indeed, strains may spend the majority of time within the community they were injected in, thus avoiding strains injected in other communities. Fig 3D confirms this hypothesis by showing the Inverse Participation Ratio (IPR) [55] that quantifies uniformity in the repartition of abundance across communities. Values close to zero indicate uniform repartition, while, conversely, values close to 1 indicate that, on average, a strain is confined within a single community for most of the time (more details are reported in the Materials and methods section). The strength of the community structure does not affect the repartition of the total prevalence (squares in the plot), however it alters the average IPR value computed from the abundance of single strains, thus strains become more localized as pIN increases. Notice that a certain degree of localization is present also in the homogeneous network, due to those strains causing very few generations before going extinct. As a sensitivity analysis we tested whether the main results obtained so far are the same in a more realistic situation where additional heterogeneous properties of nodes are accounted for. We consider the case in which infectious duration varies across individuals, as happens for S. aureus colonization. S7 Fig shows that the inclusion of three classes differing in recovery rate reduces richness and increases the Berger-Parker index with respect to the homogeneous recovery. However, the effects discussed so far—e. g. reduction and amplification of richness in HET and COM, respectively—are still present. Node turnover represents another important property of a network that may impact the ecological dynamics of strains for two reasons: incoming individuals contribute to richness by injecting new strains; on the other hand, the removal from the population of infected nodes breaks transmission chains and hampers the persistence of strains. The result of the interplay between these two mechanisms is summarized by the plot of richness as a function of β and node length of stay, τ, —Fig 4A. The figure, obtained with the HOM model, shows two distinct regimes. In the former case, richness decreases as τ increases, because replacement of individuals becomes slower and injections less frequent. In the high β regime, instead, the average richness at fixed β does not depend monotonically on the node turnover but it is instead maximized at intermediate τ. Interestingly, the optimal value of τ decreases as β increases. This behavior can be explained by looking at the balance between injection and extinction that determines the equilibrium value of richness, N ¯ S. This reads [56]: N ¯ S = λ inp s T pers (β, τ) = V ¯ p s T pers (β, τ) τ, (1) where λinps is the rate at which new strains are introduced and Tpers is the average persistence time of a strain. The trade-off between injection and extinction appears as the ratio between the two time scales, Tpers and τ. In the limit τ → 0 the spread plays no role, even for high β. As τ increases, newly introduced infectious seeds have a higher probability to spread, thus the average extinction time initially increases super-linearly with τ (see S8 Fig in the supporting information) resulting in an increase of richness. However, past a certain value of τ, Tpers does not grow super-linearly anymore, thus a further increase in τ is detrimental for pathogen diversity because it is associated to fewer introductions. This general behavior was not altered by the accounting for introductions by transmissions from an external source as shown in S6 Fig. We derive an approximate formula for Tpers considering an emerging strain competing with a single effective strain formed by all other strains grouped together. This formulation, enabled by the neutral hypothesis, makes it possible to write the master equation describing the dynamics and to use the Fokker-Planck approximation to derive persistence times (see Materials and methods section). Analytical results well reproduce the behavior observed in the simulations, and, in particular, the value of the length of stay maximizing richness for different β as shown by the comparison between white stars and continuous line in Fig 4B. The quantitative match for other values of ps is reported in S9 Fig. Unlike richness, Berger-Parker index always increases monotonically with the length of stay—Fig 4B. This behavior is due to the correlation of this indicator with average abundance, similarly to what we discussed in the previous section. We conclude by analyzing the real-case example of the S. aureus spread in a hospital setting [10,57]. We used close-proximity-interaction (CPI) data recorded in a long-term health-care facility during 4 months by the i-Bird study [16,28,31]. These describe a high-resolution dynamical network whose complex structure reflects the hospital organization, the subdivision in wards and the admission and discharge of patients [58]. Together with the measurements of contacts, weekly nasal swabs were routinely performed to monitor the S. aureus carriage status of the participants and to identify the spa-type and the antibiotic resistance profile of the colonizing strains. The modeling framework considered here well applies to this case. The SIS model is widely adopted for modeling the S. aureus colonization [59,60], and the assumption of mutual exclusion is made by the majority of works to model the high level of cross-protection recognized by both epidemiological and microbiological studies [61,62]. The dynamic CPI network was previously shown to be associated with paths of strain propagation [16]. Consistently, we assumed that transmission is mediated by network links with transmissibility β. In addition, new strains are introduced in the population carried by incoming patients, or through contacts with persons not taking part in the study. Fig 5A shows weekly carriage and its breakdown in different strains. Prevalence and richness fluctuate around the average values 87,3 ± 6,3 cases and 39,8 ± 2 strains, respectively. Simulation results are reported in Fig 5B, that displays the impact of transmission and introduction rate on richness and prevalence. When qs is low we find a positive trend between richness and prevalence, consistently with the synthetic case. For larger values of qs the trend appears instead different. As transmissibility increases, richness initially grows with prevalence and then decreases after a certain point. This behavior is the same as observed in S6 Fig and stems from the reduction of susceptible nodes, that causes a decline in the expected injection rate—see Materials and methods section. To quantify the effect of contact patterns on S. aureus population ecology we compared simulation results with the ones on a network null model. Specifically, we built the RAND null model that randomizes contacts while preserving just the first and the last contact of every individual. The randomization preserves node turnover, the number of active nodes and links and destroys contact heterogeneities and community structure along with other correlations. Fig 5C shows the comparison for different transmissibility values. The effect of the network is consistent with the theoretical results described for a heterogeneous network, i. e. smaller richness values correspond to the same prevalence in the real network compared to the homogeneous one. We then quantified the level of dominance of the multi-strain distribution by means of the Berger-Parker index. We chose for each network the values of qs and β that better reproduce empirical richness and prevalence and, interestingly, we found that, for the two cases, same average richness and prevalence correspond to different levels of Berger-Parker index. The Berger-Parker index obtained with the real network is the highest and the one that better matches the empirical values—i. e. the empirical values are within one standard deviation of the mean for almost all weeks. Based on this result we argue that contact heterogeneities, along with the other properties of the contact network, contribute to the increased dominance of certain strains. Multiple biological and environmental factors concur in shaping pathogen diversity. We focused here on the host contact network and we used a minimal transmission model to assess the impact of this ingredient on strain population ecology, quantifying the effects of three main network properties, i. e. heterogeneous activity potential, presence of communities and turnover of individuals. Results show that the structure and dynamics of contacts can alter profoundly strains’ co-circulation. Contact heterogeneities were found to shape the distribution of strains’ abundances. Highly active nodes are known to play an important role in outbreak dynamics by acting as super-spreaders [33]. At the same time, however, they were found to enhance the interference between the transmission chains of different strains, thus hindering the spread of an emerging variant [46]. Here we showed that the combination of these two dynamical mechanisms reduces the richness and increases the level of heterogeneity in strains’ abundances. In particular, hubs could allow strains with no biological advantage to generate a large number of cases and outcompete other equally fit strains. This mechanism may potentially bias the interpretation of biological data. Dynamical models that do not properly account for contact structure could overestimate the difference in strains’ epidemiological traits in the attempt to explain observed fluctuations in strain abundance induced in reality by super-spreading events. Moreover, these models could provide biased assessment of transmission vs. introduction rates. The presence of communities causes the separation of strains and mitigates the effect of competition thus enhancing co-existence. A similar behavior was already pointed out before [46,51,59,64], e. g. for the spread of S. pneumoniae, as induced by age assortativity [64], for the case of S. aureus where distinct settings were considered [59], and for a population of antigenic distinct strains in presence of cross-immunity [51]. We found that the impact of community structure is not so strong, and it is likely minor when individuals of different communities have frequent contacts. No appreciable variation was observed, indeed, for pIN = 0. 78, chosen to match the inter-ward coupling of the hospital network. Similar results can be expected for school classes or workplace departments presenting a similar level of community mixing. The effect on richness becomes appreciable for low community coupling (e. g. pIN = 0. 99 in Fig 3). This is consistent with a certain degree of diversity observed among strains belonging to separated communities, as it is the case of different hospitals [15]. Eventually, the analysis of turnover of individuals revealed major effects on strain diversity, when this mechanism is also the main driver of the introduction of strains in the population. When transmissibility is low richness decreases with host length of stay. When transmissibility is above the epidemic threshold we showed the existence of an optimal value of the length of stay that maximizes strain richness as a result of the interplay between two competing time scales, namely the typical inter-introduction time and the average persistence time of a strain. This provides insights for the spread of bacterial infections in transmission settings, such as hospitals or farms, that are of particular relevance for the spread of antimicrobial resistance and that are characterized by a rapid host turnover [15,31,65]. For the case of hospitals, for instance, they suggest that variations in patients’ length of stay, as induced by a change of policy, could have appreciable effects on the population structure of nosocomial pathogens. We adopted a neutral model to better disentangle the relative role of the different network properties. A wide disease-ecology literature addressed the consequences of neutral hypotheses on multi-strain balance in order to provide a benchmark for interpreting the observed co-existence patterns and gauging the effect of selective forces potentially at play [11,18,66,67]. Many of these works addressed, for instance, the co-existence between susceptible and resistant strains of S. pneumoniae [11,66]. However, this assumption was rarely adopted in network models, that consider for the majority strains with different epidemiological traits with the aim of describing pathogen selection and evolution [47–49,68]. Strains were assumed to have the same infection parameters in [50,51], where the role of community structure and clustering was analyzed in conjunction with cross-immunity. With respect to these works, the minimal transmission model used here enabled a transparent comprehension of the role of the network. Multiple identical SIS processes can be mapped, in fact, on a single SIS process, in such a way that the wide literature of single SIS processes allows for a better understanding of the behavior recovered in the simulations [32,33]. Strains can be also grouped in two macro-strains. This strategy allowed us to adopt the viewpoint of an emerging strain and study its competition with the others seen as a unique macro-strain. The associated master equation and Fokker-Planck approximation allowed computing the average extinction time, capturing the key aspects of the dynamics. In a future work this theoretical framework could be extended to consider other network topologies. It could, for instance, be coupled with the activity-block approximation to describe heterogeneous networks. Additional numerical analyses, based on a similar transmission model, could also address other properties known to alter spreading dynamics, such as heterogeneous inter-contact time distribution or topological and temporal correlations. As a case study, we analyzed the spread of S. aureus in a hospital taking advantage of the simultaneous availability of contact and carriage information [16]. The temporal and topological features of the network lead to a lower prevalence and richness with respect to the homogeneous mixing (although the effect was quite small). In addition, similar prevalence and richness values are associated to different dominance levels in different networks—i. e. different values of the Berger-Parker index—with the real network leading to a higher dominance as observed in reality. This behavior can be explained by the theoretical results and can be attributed essentially to the effect of contact heterogeneities, considering that the community structure does not have appreciable effects for this network, as discussed above. The importance of accounting for host contacts and hospital organization in the assessment of bacterial spread and designing interventions has been recognized by several studies [16,28–31,63]. Here we show that this element may be critical also for understanding the population ecology of the bacterium. It is important to note however that, while the realistic network provides results that are closer to the data, this ingredient explains only part of the heterogeneity observed in the abundance. This shows that the contact network is a relevant factor, but other factors should be considered as well. The approach used here is intentionally simplified, as we focused on the main dynamical consequences of the contact network. Clearly, more detailed models can be designed to reproduce more closely the data. A certain degree of variation in the epidemiological traits could be at play, as for example the fitness cost of resistance [8]. Role of hosts in the network (e. g. patients vs. health-care workers), and heterogeneities in health conditions, antibiotic treatment and hygiene practices are also known to affect duration of carriage and chance of transmission [16,28,31,63]. Eventually, we must consider that the comparison of model output with carriage data is also affected by the limitation of the dataset itself, already described in [16]. In particular, the weekly swabs may leave transient colonization undetected. Moreover, while the relevance of CPIs as proxies for epidemiological links has been demonstrated [16], the transmission through the environment (e. g. in the form of fomites) is also possible. The understanding provided here can be relevant for other population settings, temporal scales and geographical levels. In addition, the modeling framework could be applied to pathogens other than S. aureus, such as human papillomavirus, S. pneumoniae and Neisseria meningitidis, for which the strong interest in the study of the strain ecology is justified by the public health need for understanding and anticipating trends in antibiotic resistance, or the long-term effect of vaccination [1,2, 4,5]. With this respect, if the simple framework introduced here increases our theoretical comprehension of the multi-strain dynamics, more tailored models may become necessary according to the specific case. In particular, we have considered complete mutual exclusion as the only mechanism for competition. In reality, a secondary inoculation in a host that is already a carrier may give rise to alternative outcomes, such as co-infection or replacement [69]. In addition, infection or carriage may confer a certain level of long-lasting strain-specific protection and/or a short-duration transcendent immunity [11,50]. Eventually mechanisms of mutation and/or recombination are at play and their inclusion into the model can be important according to the time scale of interest. We provide here details of the generative algorithms used for the contact network models. Network dynamics is implemented in discrete time according to the following rules common to all models: Turnover dynamics: new nodes arrive according to a Poisson process with rate λin and leave after a random time drawn from an exponential probability distribution with average τ. After a short initial transient, population size is Poisson distributed with average V ¯ = λ in τ. Upon admission, a node i is assigned with an activity potential ai, i. e. an activation rate, drawn at random from a given probability distribution P (a). Any node retains this property throughout its whole lifespan. Activation Pattern: each node i becomes active with rate ai. It then receives a number of stubs drawn from a zero-truncated Poisson distribution with parameter κ—we require active nodes to engage in at least one contact. The average number of stubs, computed among active nodes, is thus given by κ/ (1 − e−κ), and the average degree can be computed by the latter quantity multiplied by the average activity potential. The active status lasts for a single time step. Stub-matching: stubs are then matched according to the actual model considered. We now describe in detail each network model: HOM: in this model each node has the same probability aH to be active during each time step; the activity distribution is thus P (a) = δ (a − aH), where δ (x) is the Dirac’s delta function. Stubs are matched completely at random in order to form links, according to a configuration model [33]. We discard eventual self-links and multiple links that may occur during the matching procedure. HET: here each node i has its own activity rate ai, drawn from a power-law distribution P (a) ∝ a−γ, with a ∈ (ϵ, 1]. We tune the variance by varying γ—lower γ higher variance. We then set ϵ to have the average activity a ¯ equal to aH in HOM. Stub-matching procedure is the same as in HOM. HET model is thus a variant of the activity driven model introduced in [34] with the difference that here contacts are created only among active individuals. COM: incoming nodes are assigned to one among nC communities with equal probability—so that communities have the same size on average—and belong to the same community throughout their whole lifespan. Stubs are matched according to the community each node belongs to. Precisely, any stub is matched either with another stub of the same community, with probability pIN, or with a stub of a different community, with complementary probability. Here the stub-matching procedure results in a larger number of lost links—to eliminate multiple links and self-loops—compared to HOM and HET, due to the difficulty in matching stubs within small groups. Thus, the parameter κ has to be adjusted manually to recover the same average degree as in HOM and HET. Each node has the same activity potential aH as in HOM. We use a dynamical contact network obtained from CPI data collected during the i-Bird study in a French hospital. Details of the network are already reported in [16]. Briefly, the dataset describes contacts occurring between 592 individuals from July to November 2009. The study involved both patients and health-care workers, distributed in 5 wards, as well as hospital service staff. Every participant wore a wireless device designed to broadcast a signal every 30 s containing information about its ID. Signal strength was tuned so that only devices within a small distance (around 1. 5 m) were able to register a contact. CPIs were finally aggregated daily, keeping the information about their cumulative duration within each day. We discard CPIs relative to the first 2 weeks and the last 4 weeks of dataset, corresponding to a period of adjustments in the measurements and progressive dismissal of the experiment, respectively. Simulations conducted with the CPIs network were compared with results obtained with a null model which we refer to as RAND. According to this randomization scheme the activity of a node is randomized while respecting the constraint that removal and addition of contacts must not alter the time of the first and the last contact of each node (tS and tL respectively). Notice that RAND preserves the number of nodes that are present at any time in the network by preserving their first contact tS and their length of stay tL − tS. Null models randomizing the latter properties lead to misleading results when node length of stay is heterogeneous and node turnover occurs [70]. RAND also sets all contact weights equal to the average weight value. Spreading dynamics is stochastic and is performed in discrete time. At each time step of duration Δt, we update the state of each node: each infected node transmits the strain it is carrying to a susceptible neighbor with probability βΔt and it turns susceptible with probability μΔt. Notice that due to mutual exclusion, an individual can be infected by a single strain at a time [71]. Strain injection is given by the combination of two processes: incoming individuals bring a new strain with probability ps, and susceptible individuals turn infectious with a new strain with probability qsΔt. The two mechanisms mimic respectively incoming infectious individuals (e. g. admission of colonized patients) and transmission from an external source (in the hospital example this corresponds to contacts with individuals that were not participating in the study). The expected injection rate, which accounts for both introduction mechanisms, is thus given by ι = λ in p s + S ¯ q s, where S ¯ is the average number of susceptible individuals at the equilibrium. In the theoretical analysis in the main paper we assumed qs = 0 for simplicity, thus variations in ι were induced by variations in λin and ps. The case qs > 0 was considered in the supporting information. Simulations on synthetic networks differ from those on the hospital network in the combination of the spreading and network dynamics. In the synthetic network case, at each time step of duration Δt = 1h, both network and spreading dynamics are simulated one after the other. On average, λinΔt new nodes enter in the population per time step, while existing nodes can leave with probability Δt/τ. Nodes then form contacts according to the specific generative network algorithm. Eventually, transmission and recovery are simulated as explained above. In order to reconstruct the equilibrium dynamics we run simulations for a sufficiently long time span, discarding a transient time of 4 ⋅ 104 time steps. We verified that the dynamical properties at the equilibrium are unaffected by initial conditions. For the hospital example, the network is an external parameter fed into the simulations. Contacts were aggregated daily keeping the information of their total duration. We used this information by considering a weighted network with the link weight, wij, representing the number of contacts of duration 30 s registered during the day between i and j. We then assumed Δt = 1 day and computed the probability of infection depending on the weight as 1 − (1 − β δ) w i j, with δ = 30 s. We initialized the system with the same configuration observed in the data, i. e. the initial status for each node is set according to S. aureus carriage during the starting week. Simulation length is bound to the hospital contact network duration. In order to facilitate the comparison between the synthetic and the real scenarios, parameters of the network models were set based on the properties of the hospital network. The average size, the average activity potential and the average degree were set equal to the values estimated from the hospital network, i. e. V ¯ = 306, a ¯ = 0. 28, k ¯ = 0. 89 respectively. For the COM model the number of communities (nC = 6) and one of the two explored values of pIN (pIN = 0. 78) were also informed by the data. Additional values of V ¯ and pIN were also tested. Epidemiological parameters were informed by the data in some cases—ps = 0. 079 as computed from carriage data -, or chosen among plausible values for the S. aureus colonization—i. e. μ−1, that was set equal to either 21 or 35 days with other values from 14 to 49 days explored in the supporting information. Values of β were explored systematically. For consistency, values of rates throughout the manuscript were always expressed per hour. Carriage data was obtained from weekly swabs in multiple body areas, including the nares. Swabs that resulted positive to S. aureus were further examined. Spa-type and antibiotic resistance profiles (MSSA or MRSA) were then determined. In this work we regard two strains as different if they differ in spa-type and/or antibiotic resistance profile. We considered carriage data obtained from nasal swabs dismissing other body areas since the anterior nares represent the most important niche for S. aureus [72]. We described strain population diversity through standard ecological indicators. The abundance of a strain i, Ni, is the strain-associated prevalence. From this quantity we computed the relative abundance, f i = N i ∑ i N i, and the relative abundance distribution, being the frequency of strains with relative abundance f. The Berger-Parker index is the relative abundance of the dominant strain, i. e. maxi fi. To analyze repartition of strains across communities we use the Inverse Participation Ratio (IPR) [55]. The general definition of this quantity is the following. Given a vector v → with l components {vi}i=1, …, l, all within the range [0,1], the IPR is given by: I P R = ∑ i = 1 l v i 4. (2) If all the components are of the order (l−1) then the IPR is small. Instead if one component vi ∼ 1 then IPR ∼ 1 too, reflecting localization of v →. The IPR for total prevalence is computed by setting vi equal to the fraction of infected individuals belonging to community i = 1, …, l = nC, while the IPR for a single strain is computed by setting vi equal to the fraction of individuals infected by that particular strain and belonging to community i. We can extend the IPR computation to HOM case by assigning nodes to different groups as in COM but without affecting the stub-matching scheme. In order to estimate the value of the length of stay maximizing the average richness for a given value of β when the contact structure is given by the HOM network we consider a homogeneous mixing version of our system. Due to Eq (1) the calculation of the average richness reduces to the calculation of the average persistence time. In order to estimate such quantity we focus on a particular strain, labelled as “strain A”, which is injected at t = 0 and we group all other strains under the label “strain B”. We are allowed to do so because all strains have identical parameters. We therefore reduce our initial, multi-strain problem, to a two-strain problem. Since all new strains that will be injected after t = 0 will be labeled as strain B, it is clear that A is doomed to extinction since there exists an infinite reservoir of B. The average time to extinction is therefore the average time to extinction of strain A. Since HOM network realizes quite well homogeneous mixing conditions we regard our system as homogeneously mixed. Within this framework it is sufficient to specify the numbers of hosts infected by strain A (nA), hosts infected by strain B (nB) and susceptible hosts (ns). The master equation for the joint probability distribution P (nA, nB, ns) is given by [73]: P ˙ (n A, n B, n s) = β ′ V ¯ − 1 (n A − 1) (n s + 1) P (n A − 1, n B, n s + 1) + β ′ V ¯ − 1 (n B − 1) (n s + 1) P (n A, n B − 1, n s + 1) + μ (n A + 1) P (n A + 1, n B, n s − 1) + μ (n B + 1) P (n A, n B + 1, n s − 1) + λ o u t (n A + 1) P (n A + 1, n B, n s) + λ o u t (n B + 1) P (n A, n B + 1, n s) + λ o u t (n s + 1) P (n A, n B, n s + 1) + λ o u t V ¯ p s P (n A, n B − 1, n s) + λ o u t V ¯ (1 − p s) P (n A, n B, n s − 1) − [ (n A + n B) (β ′ V ¯ − 1 n s + μ) + λ o u t (n A + n B + n s) + λ o u t V ¯ ] P (n A, n B, n s), (3) Where β ′ = β k ¯. Terms appearing on the right-hand side of the equation represent the probability flow associated to each transition event. The first four terms describe, in order, the infection due to strain A, the infection due to strain B, the recovery from A and the recovery from B. The remaining terms are then associated to the discharge of either one of the three types of individuals—infected with A, infected with B and susceptibles—and to the admission of infected of type B and susceptibles respectively. In order to obtain some approximate solution to this equation we assume that the average number of individuals nA + nB + ns and the total prevalence nA + nB do not fluctuate in time and are therefore equal to V ¯ and i (∞) V ¯ respectively, where i (∞) is given by: i (∞) = β ′ − μ − λ o u t + (β ′ − μ − λ o u t) 2 + 4 β ′ λ o u t p s 2 β ′. (4) After performing the Van-Kampen size expansion we are left with a Fokker-Planck equation for the density of A f (x = n A V ¯) = P (n A): ∂ t f = − ∂ x (D 1 (x) f) + 1 2 V ¯ ∂ x 2 (D 2 (x) f), (5) where D1 = β′ (1 − i (∞) ) x − μ − λout and D2 = β′ (1 − i (∞) ) x + μ + λout are the so-called drift and diffusion coefficients respectively. According to the theory of stochastic processes [73] the average extinction time Tpers (x0) (where x0 represents the initial density of strain A) satisfies: D 1 (x 0) d d x 0 T pers + 1 2 V ¯ D 2 (x 0) d 2 d x 0 2 T pers = − 1, (6) with boundary conditions Tpers (0) = 0 and d d x 0 T pers (i (∞) ) = 0. The solution is finally given by: T pers (x 0) = i (∞) λ o u t p s [ E i (− α i (∞) ) (e α x 0 − 1) − e α x 0 E i (− α x 0) + l n (α x 0) + γ E ], (7) where Ei (x) is the exponential integral function and γE is Euler-Mascheroni constant. When a new strain is introduced its prevalence is just 1, therefore we estimate the average extinction time using T pers (x 0 = V ¯ − 1). | Title: Host contact dynamics shapes richness and dominance of pathogen strains Summary: Pathogens are structured in multiple strains that interact and co-circulate on the same host population. This ecological diversity affects, in many cases, the spread dynamics and the efficacy of vaccination and antibiotic treatment. Thus understanding its biological and host-behavioral drivers is crucial for outbreak assessment and for explaining trends of new-strain emergence. We used stochastic modeling and network theory to quantify the role of host contact behavior on strain richness and dominance. We systematically compared multi-strain spread on different network models displaying properties observed in real-world contact patterns. We then analyzed the real-case example of Staphylococcus aureus spread in a hospital, leveraging on a combined dataset of carriage and close proximity interactions. We found that contact dynamics has a profound impact on a strain population. Contact heterogeneity, for instance, reduces strain diversity by reducing the number of circulating strains and leading few strains to dominate over the others. These results have important implications in disease ecology and in the epidemiological interpretation of biological data. | 10,525 | 248 | lay_plos | en |
Summarize: La detox water è un'acqua aromatizzata dalle proprietà drenanti e depurative che può essere preparata anche in casa. Si tratta infatti di un'acqua minerale, arricchita con frutta, spezie e verdure, lasciate in infusione e non sbucciate, che può essere bevuta durante tutto l'arco della giornata. È una bevanda rinfrescante, perfetta per la stagione estiva che avanza, ma in grado anche di regalare al nostro organismo numerosi benefici. Alle proprietà dell'acqua, infatti, i principi attivi degli ingredienti che scegliamo di inserirvi aggiungono ulteriori proprietà, da quella antiossidante a quella antinfiammatoria. Sono bevande ricche di vitamine e completamente naturali, prive di zuccheri aggiunti e piacevoli da bere. Preparare un'acqua depurativa e drenante è molto semplice. Basterà scegliere gli ingredienti che meglio rispondono alle nostre esigenze, tra erbe aromatiche, frutta e ortaggi. Alcuni, infatti, aiutano a combattere la ritenzione idrica, altri hanno effetti digestivi o permettono di stimolare il nostro metabolismo. Dopo aver scelto gli ingredienti, preferibilmente biologici e non trattati, occorrerà lavarli bene e poi inserirli all'interno di una brocca di acqua fredda. Bisognerà schiacciarli delicatamente, per poter spremerne il succo e poi lasciarli in infusione per almeno 4-5 ore circa. Maggiore sarà il tempo, più l'acqua acquisterà sapore: in alcuni casi è, infatti, consigliabile preparare l'acqua la sera prima, così da lasciarla in infusione tutta la notte. Una volta pronta la nostra detox water, potremo conservarla in frigo e consumarla entro 3 giorni dalla sua preparazione. Prima di berla, possiamo scegliere di filtrarla oppure mangiare anche la frutta e gli ortaggi, così da assimilare una maggiore quantità di fibre. Ecco allora alcune ricette per preparare la nostra acqua detox. Per chi combatte ogni giorno con la sensazione di gonfiore, può preparare un'acqua detox a base di zenzero, che avrà non solo effetto antiossidante e digestivo, ma ci potrà aiutare anche a riattivare il nostro metabolismo. Tutto ciò che occorre fare è tagliare a fettine sottile lo zenzero e unirlo, all'interno di una brocca d'acqua, con del lime e del cetriolo non sbucciato, ma tagliato a pezzetti. Basterà lasciare in infusione la bevanda per alcune ore nel frigorifero. Il gusto, a causa dello zenzero, potrebbe risultare leggermente forte, ma sarà comunque rinfrescante e dissetante. Una ricetta efficace e semplice da preparare è l'acqua a base di limone e cetriolo, dall'azione drenante e depurante. Bisogna aggiungere in un litro e mezzo di acqua del limone, tagliato a fettine sottili e già ampiamente utilizzato per la sua azione detox, insieme a un cetriolo in piccoli pezzi. Il preparato aiuterà il nostro organismo anche a eliminare le tossine. In questo caso è preferibile berne due bicchieri al mattino, appena alzati e prima di fare colazione. L'arancia è un perfetto alleato del nostro organismo, poiché va a stimolare il sistema immunitario grazie all'elevata concentrazione di vitamina C. Basterà tagliarla a spicchi e inserirla in una brocca contente un litro e mezzo d'acqua, insieme a un limone e a un rametto di rosmarino. È consigliabile lasciarla in infusione per cinque ore almeno. Come ricetta alternativa, possiamo aggiungere all'acqua tre arance tagliate a spicchi, mezzo limone spremuto e qualche foglia di salvia. In questo caso la nostra bevanda potrà essere utilizzata come digestivo o come naturale antinfiammatorio. Per realizzare una detox water dall'azione antiossidante, gli ingredienti migliori da utilizzare sono mele, kiwi e limoni. Bisogna aggiungere in un litro e mezzo d'acqua circa delle fettine di mela e di limone, entrambi con la buccia, e dei pezzetti di kiwi privi ovviamente della loro pelle. La mela aiuta nella digestione e ha una buccia ricca di vitamine, il limone permette di bruciare i grassi ed è antiossidante come il kiwi, ricco di vitamina C ed E, rame, ferro e potassio. | Summary: L'acqua detox è un infuso completamente naturale e privo di zuccheri aggiunti o conservanti, che possiamo preparare con facilità e bere durante queste giornate che ci avvicinano alla calda stagione estiva. Si tratta di una bevanda che utilizza i principi attivi di frutta, spezie e ortaggi per regalare al nostro organismo una serie di benefici. Ecco allora cinque ricette per preparare detox water dalle proprietà antiossidanti, antinfiammatorie e abbronzanti. | 894 | 96 | fanpage | it |
Write a title and summarize: Nuclear receptors of the Hepatocyte Nuclear Factor-4 (HNF4) subtype have been linked to a host of developmental and metabolic functions in animals ranging from worms to humans; however, the full spectrum of physiological activities carried out by this nuclear receptor subfamily is far from established. We have found that the Caenorhabditis elegans nuclear receptor NHR-31, a homolog of mammalian HNF4 receptors, is required for controlling the growth and function of the nematode excretory cell, a multi-branched tubular cell that acts as the C. elegans renal system. Larval specific RNAi knockdown of nhr-31 led to significant structural abnormalities along the length of the excretory cell canal, including numerous regions of uncontrolled growth at sites near to and distant from the cell nucleus. nhr-31 RNAi animals were sensitive to acute challenge with ionic stress, implying that the osmoregulatory function of the excretory cell was also compromised. Gene expression profiling revealed a surprisingly specific role for nhr-31 in the control of multiple genes that encode subunits of the vacuolar ATPase (vATPase). RNAi of these vATPase genes resulted in excretory cell defects similar to those observed in nhr-31 RNAi animals, demonstrating that the influence of nhr-31 on excretory cell growth is mediated, at least in part, through coordinate regulation of the vATPase. Sequence analysis revealed a stunning enrichment of HNF4α type binding sites in the promoters of both C. elegans and mouse vATPase genes, arguing that coordinate regulation of the vATPase by HNF4 receptors is likely to be conserved in mammals. Our study establishes a new pathway for regulation of excretory cell growth and reveals a novel role for HNF4-type nuclear receptors in the development and function of a renal system. Nuclear receptors (NRs) comprise a large family of transcription factors distinguished by a highly conserved DNA binding domain and a structurally conserved ligand-binding domain. NRs are notable for their ability to interact with small molecule ligands, enabling these factors to respond to autocrine, paracrine, and endocrine signals in order to mediate transcriptional effects at a distance [1], [2]. The canonical NR family is exclusively found in metazoans and the number of nuclear receptor members varies dramatically depending on species; from 21 NR genes in Drosophila melanogaster, to ∼50 in rodents and humans, to over 250 NRs in Caenorhabditis elegans and related nematodes [3]. The extraordinarily large NR family of C. elegans is particularly intriguing. Of the 283 predicted NR genes, only 15 are directly orthologous to NRs found in other metazoans, including Drosophila and mammals [4]. The remaining 268 NRs are thought to be derived from extensive duplication and diversification of an ancestral gene most closely related to the mammalian and Drosophila HNF4 receptors [5]. The presence of both highly similar and divergent HNF4-type receptors in nematodes implies that many of these proteins will carry out conserved structural and physiological functions, whereas others will have evolved to adopt responsibilities more specific to the nematode lineage. This idea is supported by the fact the C. elegans NHR-49 nuclear receptor shares many of the metabolic functions of the mammalian HNF4α, but not the developmental activities [6], [7]. Thus, study of C. elegans NRs should not only be helpful for understanding mammalian NR function and physiology, but should also reveal novel regulatory activities for the nuclear receptor family. The prospect that the responsibilities of mammalian receptors may be divided among a larger number of NRs in C. elegans may be advantageous for understanding the physiological function these complex proteins. For example, the mammalian HNF4α plays numerous roles in development, metabolism, and disease [8]; because of this widespread physiological impact, the functional and mechanistic diversity of this receptor is far from understood. Indeed, mutations in the human HNF4α are associated with maturity onset diabetes of the young (MODY) and late onset type II diabetes; yet, how these HNF4α lesions lead to diabetes has not been established [9]–[11]. Furthermore, there is considerable controversy over the quantity and identity of HNF4 target genes [12]–[14]. These complications may be due, at least in part, to the fact that HNF4α carries out essential functions in several different tissues, and that HNF4α likely regulates different target sets depending on metabolic, developmental, and nutritional context. HNF4α is also expressed in many cell types for which its function has not yet been established; for example, the epithelial cells of the intestine and the proximal and convoluted tubules of the kidney, and while HNF4α has been shown to regulate proliferation of transformed kidney cell lines, its role in kidney development remains to be defined [15], [16]. The C. elegans renal system is comprised of only three cells, yet these cells carry out many of the same functions as mammalian kidneys [17], [18]. Therefore, C. elegans might be an advantageous system in which to study the role of HNF4 receptors in renal development. The largest portion of the C. elegans excretory system consists of the excretory cell (EC). The development of the EC is extraordinary, as it involves the formation and growth of four branches that project outward from a single nucleus located near the anterior bulb of the pharynx [17]. These branches grow along the length of the animal to near the tip of the head and tail in early development, and then continue to grow along with the animal until adulthood. Each branch of the EC contains an inner membrane that coalesces to form a lumen; thus, the excretory cell becomes a large, single cell tube. Consequently, the EC has been effectively used to understand the development of tubes and to investigate mechanisms involved in excretory function [17], [19], [20]. At this point, factors known to participate in the development and function of the C. elegans excretory cell include vATPases, WNK kinases, CLIC-like proteins, Patched related proteins, and mucins [17], [21]–[24]. Additionally, the CEH-6 homeobox protein has also been implicated as the only transcriptional regulatory factor, thus far, involved in excretory cell development [25]. How the complex structure of the EC is developed and maintained so precisely, even at points very distant from the primary sites of gene regulation, remains a mystery. We have found a highly conserved C. elegans HNF4 paralog, NHR-31, that is specifically expressed in the excretory cell of the nematode, suggesting that investigation of this receptor may provide unique insight into the role of nuclear receptors in renal development and tube formation. In this study, we show that NHR-31 specifically regulates the expression of genes that coordinate the synchronous growth and elongation of excretory canals, demonstrating a novel NR mediated pathway for renal system development and function. nhr-31 is predicted to encode an HNF4α related nuclear receptor (NR) protein with a highly conserved DNA binding domain (DBD) and ligand binding domain (LBD) (Figure 1A). To help establish the physiological function of this NR, we determined the tissues in which the nhr-31 gene is expressed. A GFP reporter construct was generated by fusing 3. 0 kb of nhr-31 upstream regulatory sequence to the gfp gene (Pnhr-31: : gfp). Injection of Pnhr-31: : gfp into WT worms revealed that the nhr-31 promoter drives strong expression in the excretory cell (EC). In transgenic animals, GFP protein was first observed in the EC cell shortly after EC birth and persisted in the EC for the remainder of worm embryogenesis, larval development, and adulthood (Figure 1B and data not shown). GFP was observed throughout the cytoplasm of the H-shaped excretory cell. Because our reporter construct was designed by fusing only the nhr-31 promoter to the gfp gene, the GFP localization pattern does not represent NHR-31 protein sub-cellular localization. Pnhr-31: : gfp expression was also observed, at lower levels, in the intestine and in several unidentified cells located near the tail (Figure 1B and data not shown). In C. elegans, the EC functions cooperatively with duct and pore cells, and together these cells are important for maintaining osmolarity homeostasis [26], [27]. To determine if nhr-31 RNAi animals displayed compromised excretory function, we treated animals with nhr-31 RNAi or control RNAi from the L1 to L4 stage of development and then stressed L4 animals with acute exposure to a standard growth plate supplemented with 500 mM NaCl, and determined their ability to respond to these unfavorable conditions. 250 animals were assayed at each time point. After just two hours, less than 5% of nhr-31 RNAi animals could be rescued from 500 mM NaCl exposure. In contrast, L4 animals fed control RNAi were able to thrive for much longer under these same conditions, with over 50% of animals maintaining the ability to recover even after 8 hours of high salt exposure (Figure 1C). These data indicate that reducing nhr-31 gene expression strongly impairs the ability to survive acute osmotic stress. Three different nhr-31 deletion strains have been isolated, and all of these strains are inviable (www. wormbase. org). Using one of these strains (nhr-31 (tm1547) ), we found that nhr-31 deletion leads to early embryonic lethality (data not shown). Additionally, application of nhr-31 RNAi throughout growth and development results in significant embryonic lethality in the F1 generation (data not shown). Thus, NHR-31, like its mammalian homolog HNF4α, plays an essential role in early embryonic development. Because we found that the nhr-31 gene is primarily expressed in the excretory cell during larval and adult stages, however, we investigated the participation of nhr-31 in EC development and morphology using an RNAi feeding strategy that specifically reduced nhr-31 expression during larval development and adulthood. In postembryonic animals, the EC is an H-shaped cell, with four canals emanating from a main cell body located near the terminal bulb of the pharynx [17]. Two canals project along each side of the animal towards the posterior end, and two canals project forward towards the anterior end (Figure 2A). To monitor EC morphology, WT animals were injected with the Pnhr-31: : gfp reporter. In WT adult animals, GFP localization revealed that the outer diameter of the excretory cell was relatively uniform through the entire length of the canal, measuring ∼3. 5 µm in proximal sections of the posterior canal, and tapering to ∼2. 4 µm in distal sections of the posterior canal (Figure 2B and 2C). When WT animals carrying the Pnhr-31: : gfp construct were treated with nhr-31 RNAi from the L1 stage of larval development through adulthood, the morphology of the adult EC was dramatically altered (Figure 2B and 2C). In particular, the excretory canals were not uniform in diameter; instead, they contained multiple enlarged varicosities, with diameters up to 10 µm (Figure 2B and 2C). These varicosities showed considerable variability in size and shape and were located along the entire length of the EC, including the proximal, middle, and distal portions of the posterior arms, as well as in the anterior branches of the EC canal (Figure 2B and 2C and data not shown). DIC images of nhr-31 (+/−) heterozygotes also revealed similar excretory cell abnormalities, providing support for the specificity of our nhr-31 RNAi construct (Figure S1). High magnification of the GFP images obtained in nhr-31 RNAi animals suggested that the varicosities consisted of dense cellular material with an abundance of vacuoles (Figure 3A). This phenotype was different from previously reported EC abnormalities, which showed enlargement of the EC cell due to fluid accumulation or cyst formation [19], [27]. To more closely examine the morphological defects in the EC of nhr-31 RNAi animals, we employed high pressure freezing transmission electron microscopy (HP-TEM). Table 1 shows quantitative analysis of sections obtained from the middle region of the EC in 5 different control RNAi animals and 5 different nhr-31 RNAi animals. Cross sections of the EC of a WT animal showed a single circular lumen with an average diameter of 1. 6 µm (Table 1). Additionally, an abundance of well-formed canaliculi were clearly visible in WT animals (Figure 3C and Table 1). Canaliculi are smaller “mini-canals” surrounding the canal lumen; these canals are thought to greatly increase the apical surface area of the EC lumen (Figure 3B) [17]. Canaliculi were visible in the wild type excretory canal cross section as small, round, circular shapes and were regular in size and consistent (∼70/section) in number from section to section (Figure 3D and Table 1). According to our EM measurements, the average diameter of the EC was ∼2. 8 µm, which agreed nicely with our GFP measurements (Figure 2C and Table 1). HP-TEM imaging revealed multiple morphological defects in the excretory canals of nhr-31 RNAi animals, particularly in the varicosities (Figure 3D and Table 1). First, the average canal diameter increased to 5. 8 µm, with larger varicosities displaying diameters of up to 8 µm, and the narrow regions showing diameters from 2–3 µm. Second, the average diameter of the lumen in nhr-31 RNAi animals was increased by 26% to 1. 95 µm, and the lumen often appeared multi-lobed. The diameter of the lumen correlated strongly with the outer cell diameter, as the largest lumen diameter measurements were found within large varicosities (Table 1). Third, we found that the canaliculi were uncharacteristically irregular in size and present at much higher numbers (∼126/cell) in nhr-31 RNAi animals (Figure 3D and Table 1). Finally, the varicosities of nhr-31 RNAi animals possessed an unusually high number of large vesicles, elevated endoplasmic reticulum abundance, and a considerable increase in mitochondria (Figure 3E and Table 1). Importantly, the TEM cross sections showed that the varicosities were not a result of an EC canal lumen that was folded back on itself or bent away from the normal lateral alignment, or due to osmotic “swelling”, both of which have been previously reported for mutants that affected EC structure [19], [27]. Consequently, the EC phenotypes resulting from loss of nhr-31 function are different from previous observations and suggest that nhr-31 defects are distinctive in their mechanism of origin. In summary, both fluorescence confocal microscopy and TEM showed that loss of nhr-31 function leads to significant defects in EC canal size, shape, and microstructure. The abundance of cellular material and organelles, along with significant structural abnormalities, implies that the abnormal varicosities observed in adult nhr-31 RNAi animals are likely to result from regions of uncontrolled cellular growth. We next applied gene expression profiling to establish downstream regulatory targets of nhr-31. Gene expression was measured using C. elegans oligomer based microarrays. We carried out this study in L4 larvae, as this is the larval stage at which the EC morphology differences between WT animals and nhr-31 RNAi animals first begin to show. Overall, we found that, in nhr-31 RNAi worms, the expression of 20 genes was suppressed by greater than 2-fold and the expression of 63 genes were enhanced by greater than 2-fold (Table S1). The most striking outcome of our microarray experiments was the discovery that RNAi of nhr-31 dramatically affected the expression of 15 genes that encode subunits of the vacuolar ATPase (gene names are referred to as vha), and one gene predicted to code for a vATPase cofactor (gene name, R03E1. 2). In fact, of the 30 genes most strongly reduced by inhibition of nhr-31,15 of these were vha genes (Table S2). The vacuolar ATPase (vATPase) is an ATP-dependent proton pump, which transports protons across cellular membranes (Figure 4A). Each C. elegans vha gene encodes for one subunit of the holoenzyme, and there are 15 separate subunits that make up the holoenzyme. For several of the vATPase subunits, C. elegans possesses multiple gene isoforms; consequently there are 18 vha genes in total. As a secondary confirmation of the microarray data, we employed quantitative RT-PCR to specifically measure the mRNA levels of all 18 vATPase genes found in C. elegans. We found that the expression of 16 of these genes was reduced when nhr-31 was inhibited (Figure 4A). Importantly, previously published data show that nearly all vha subunits are expressed in the excretory cell, indicating NHR-31 is likely to be mediating expression of these vha genes directly in the EC (Table 2) [19], [20], [29]–[33]. Additionally, most vha genes are also expressed in the intestine, where NHR-31 also resides. Accordingly, the only two vha genes not regulated by NHR-31, vha-7 and unc-32, are not expressed in the excretory cell. In sum, our microarray and QRT-PCR convincingly demonstrate that a primary function of NHR-31 is to coordinately promote the expression of almost the entire complement of vacuolar ATPase genes. NHR-31 localization to the excretory cell, where nearly all vha genes are expressed, also argues that NHR-31 is regulating vha genes in this cell type. Because the vacuolar ATPase subunits are highly expressed in the EC, we suspected that the impact of nhr-31 on EC development might be a consequence of vacuolar ATPase regulation. To test this hypothesis, we used RNAi feeding to specifically reduce the expression of three different vacuolar ATPase subunits: vha-5 (small a subunit), vha-8 (catalytic E subunit) and vha-12 (B subunit). Because previous studies have shown that RNAi of the vacuolar ATPase subunits leads to larval lethality, we did not apply vha or nhr-31 RNAi until the L3 stage of development. Using this approach, we found that RNAi of each of these subunits was sufficient to cause excretory canal formation defects similar to those of nhr-31 RNAi animals (Figure 4B). These results imply that the control of EC development by NHR-31 is mediated, at least in part, by its stimulation of vATPase expression. We also note that this experiment shows that knockdown of nhr-31 or vATPase expression specifically in late larval development is sufficient to cause irregular EC growth and adult varicosities. Although the large, irregular, varicosities observed in nhr-31 RNAi animals were never observed in WT adults, we did notice varicosity-like structures early in WT larval development, residing at regular intervals along the EC canal in L1 and early L2 animals (Figure 5A and Table 3). These varicosities differed from those present in nhr-31 RNAi adults in that they displayed a consistently symmetrical oval shape (Figure 5A). In L1 larvae, ∼10 of these varicosities were observed in each EC canal branch, but as worms developed the regions of the excretory cell between varicosities grew wider and the varicosities consequently decreased in prominence such that, by the late L3 stage of development, the entire length of the excretory cell possessed a diameter similar to the varicosities observed in L1 animals (Figure 5B and 5E). The presence of these growth varicosities in WT L1 larvae was confirmed by hp-TEM (Figure 5C and 5D). According to these TEM measurements, L1 varicosities displayed a diameter that was 2. 8 times that of narrow regions, and a lumen diameter that was about 2-fold larger than the narrow regions (Table 3). Additionally, the varicosity regions harbored many more canaliculi (Figure 5C and 5D and Table 3). This data implies that the varicosities may form in L1 animals and spread horizontally along the excretory cell to help increase cellular diameter, and perhaps also length. Thus, we suspected that nhr-31 RNAi animals might improperly maintain these structures such that they continue to enlarged and become irregularly shaped as animals developed into adults. However, examination of nhr-31 RNAi animals revealed no obvious signs of varicosities in the L3 stage of development, implying that knockdown of nhr-31 did not interfere with the normal dissipation of these structures during mid-larval development (Figure 5E). The varicosities that arise in nhr-31 RNAi animals first appear in the late L4 stage of development and continue to grow larger as animals grow older (Figure 5E). Consequently, the varicosities observed in adult nhr-31 RNAi animals must either occur from growth of new structures, or the reactivation and renewed growth of these original varicosities. We also note that the varicosities caused by nhr-31 loss of function continue to grow larger during adulthood, such that by day 2 of adulthood they are nearly twice as large as varicosities in early adults (Figure 5E). Nuclear receptors typically associate with complex binding motifs comprised of two hexameric half-sites [2]. These half sites may be paired in multiple orientations with various amounts of spacing, and this architecture helps determine the type of NHR that binds. To identify NREs in the promoters of the C. elegans vacuolar ATPase genes, we used the NHR-computational analysis program “NHR-scan” [34]. This program identified strong NRE candidates in nearly all of the vha promoters; 15 out of 18 vha genes harbored candidate NREs in close proximity to their transcription start site. If a vha gene was expressed as part of an operon, NREs were found near the transcription start site of the first gene in the operon. Analysis of predicted NREs showed a strong presence of an AGTTCA consensus half site (Figure 6A and Table 4). The most common repeats were an ER6 (40% of all binding sites), which is an everted repeat separated by 6 base pairs and an ER8 (27% of all binding sites), an everted repeat separated by 8 base pairs. In fact, 13 of 19 vATPase genes had at least one highly conserved ER6 or ER8 site in their promoters, while several other types of AGTTCA repeats were also found once or twice in vATPase promoters. Interestingly, we also found a consensus ER6 site in the nhr-31 promoter, implying that nhr-31 may regulate its own expression through a feedback or feed-forward mechanism. This putative regulation did not manifest in our GFP reporter studies, however, implying that self-regulation in the excretory cell is not very significant during development. The most common spacing for mammalian HNF4α receptors is a DR1 or DR2, however it would not be surprising if NHR-31 adopted a different NRE specificity, as nematode NR binding sites have likely evolved to generate NREs to help distinguish between all of the different HNF4 paralogs in C. elegans. Consistent with this notion, the NHR-31 LBD does not retain two conserved amino acids that help direct HNF4α homodimerization on DR1 and DR2 sites [35]. It is also possible, however, that everted repeats have not yet been widely characterized as HNF4α sites in other organisms. The presence of so many binding sites that closely match a consensus site is quit remarkable, especially since NHR response elements are notoriously degenerate [36]. Furthermore, nuclear receptor regulated genes often contain several conserved and cryptic NREs that are necessary for modulating expression level, consequently, there are likely to be important cryptic NREs in these promoters as well [37]. Analysis of the vATPase gene promoters from mice (Mus musculus) showed an astonishing enrichment of HNF4α binding sites (Table 4). In fact, we found highly conserved HNF4α binding sites in 10 vacuolar ATPase genes, and most of these genes harbored at least two independent HNF4α binding sites. The repeats were almost always in DR1 or DR2 configuration and the consensus half-site sequence for these sites was AG (G/T) TCA (Figure 6B), which matches the consensus site previously reported for HNF4α binding sites [38]. As with the C. elegans NREs, the enrichment of these binding sites is highly significant. Taken together, these data strongly argue that coordinate regulation of vacuolar ATPase genes by the HNF4 nuclear receptor is conserved in mammals. We should note, however, that the DR1 and DR2 elements can also bind other mammalian nuclear receptors; therefore, even though NHR-31 is most closely related to HNF4α, and expressed along with vATPases in the excretory system, the participation of other mammalian nuclear receptors in coordinate regulation of vATPase genes cannot be ruled out. Similarly, we cannot rule out the involvement of additional C. elegans NRs in regulation of nematode vATPase genes. We have identified a new pathway involved in the development of the C. elegans renal system. In summary, we have shown that the NHR-31 nuclear receptor, through promotion of vacuolar ATPase gene expression, is essential for the appropriate growth, morphology, and function of the C. elegans excretory cell. This study not only identifies a new transcriptional regulator necessary for EC development, but also establishes the specific regulatory targets that mediate its effects, and highlights potential nuclear receptor response elements. The regulatory or developmental activities carried out by NHR-31 have not yet been observed for a nuclear receptor; consequently our findings expand the physiological repertoire of the NR superfamily. A primary function of NHR-31 is to maintain the structure of the EC canal during the transition from larval development into adulthood. When exposed to nhr-31 RNAi throughout larval development, or specifically in late larval development, we observed numerous large and irregular varicosities all along the length of the posterior and anterior EC canals, these varicosities first manifested in L4 development and continued to amplify and grow several days into adulthood. As the excretory cell is involved in the regulation of ion transport and osmolarity, we considered that these varicosities might have been due to accumulation of fluid within the EC cytoplasm to create “cyst-like” structures. However, HP-TEM revealed numerous sub-cellular abnormalities within the varicosities that could not be explained by an abnormal accumulation of fluid. For example, nhr-31 RNAi dependent varicosities generally contained abnormally shaped lumens, significant increases in the number of canaliculi, ER and mitochondria, and abnormally large numbers of ectopic vesicles. These data imply that the EC varicosities are not fluid filled, but rather overdeveloped. In contrast, in the narrow regions of the nhr-31 RNAi EC, we found normal numbers of mitochondria, ER, and canaliculi, implying the majority of EC irregularities that occur in nhr-31 RNAi animals are localized to the enlarged varicosities. This excessive growth phenotype significantly differs from previously characterized excretory cell phenotypes [19], [27]. Another intriguing finding of our study is that NHR-31 has a surprisingly specific and strong impact on the expression of v-ATPase encoding genes (vha genes). The vacuolar ATPase (v-ATPase) is an ATP-dependent proton pump that is organized into a peripheral domain (V1), which is responsible for ATP hydrolysis, and an integral domain (V0), responsible for proton transport. Although it is referred to as the vacuolar ATPase, this enzyme is found in multiple intracellular membranes, including endosomes, lysosomes, Golgi-derived vesicles, clathrin coated vesicles, secretory vesicles, as well as the plasma membrane [39], [40]. vATPases are important for numerous cellular functions, including ion transport, substrate transport, acidification of vesicles and other organelles. Additionally, recent studies have shown that vATPases also play a predominant role in vesicular trafficking of the endocytic and exocytic pathways, participating directly in membrane fusion by not only providing the proper acidic environment, but also by directly forming protein complexes during the fusion process [40]. Given the diversity of vATPase functions, it seems likely that the transcription of vATPase would be precisely regulated both spatially and temporally in order to facilitate the development and function of different cell types. Although numerous factors have been shown to regulate the vATPase at the enzymatic level, our study has identified a transcription factor with a specific role in regulating vATPase expression in a tissue specific manner. In C. elegans it has been shown that vATPase subunits of either the V0 sector or the V1 sector, are important in excretory cell development and morphology [19]. In this previous study, several distinct vha subunits were knocked down early in development resulting in several defects in the hypodermis, cuticle, and excretory cell. Specifically, abnormal structures were observed in the ECs that were described as “whorls”. Because RNAi of nhr-31 leads to the reduced expression of 17 out of the 19 genes that encode vha subunits, we suspected that the role of nhr-31 in EC development may be due, at least in part, to regulation of vATPase gene expression. In support of this hypothesis, we found that larval specific knockdown of NHR-31 target genes encoding either an a subunit, an E subunit, or a B subunit of the vATPase, led to excretory cell phenotypes nearly identical to those observed in nhr-31 RNAi animals. Although varicosities found in our experiments may be related, in some fashion, to the “whorls” observed in the previous study ([19], it should be noted that the previous study focused on reduction of vATPase expression much earlier in development. In contrast, in our study, vha expression was knocked down specifically in late L3 development through early adulthood. Thus, our findings show that regulation of vATPase expression is a prominent factor in NHR-31 function. The phenotypic abnormalities observed in nhr-31 RNAi animals, combined with the predicted function of nhr-31 regulatory targets, provide several clues into how this nuclear receptor may impact the generation of a healthy EC (Figure 7). A critical component of EC development is the outgrowth of the excretory canals. During larval development, four excretory canals must grow out of the main cell body and migrate towards the posterior and anterior ends of the animal and then continue to grow as the animal increases in length. We observed that, during early larval development, the EC migrates along the length of the animal and is periodically punctuated with small oval shaped varicosities. By the time a worm reaches later larval stages, these varicosities are no longer present and adult EC canals are exquisitely uniform in diameter. We suggest that the growth varicosities that form during early larval development may be regions of high cellular growth activity, where robust protein, organelle, and membrane synthesis occur, these areas of growth then serve to supply material to the cytosol, as well as the basal and apical membranes of the EC, thus enabling the EC canal to elongate in a bidirectional manner. TEM images of the EC in L1 larvae, which show periodic varicosities with a more dense supply of membrane and organelles, support this hypothesis (Figure 5C and 5D). As the EC reaches its full-length, precise regulation of new cellular synthesis and cellular elongation reaches equilibrium such that regions of high EC cellular mass become evenly distributed and the EC adopts a fully mature and uniform shape. Many of defects observed in nhr-31 RNAi animals are consistent with an inability to properly regulate the coordination between EC cell outgrowth and new synthesis of cellular material. Thus, the NHR-31 nuclear receptor may play an important role in regulating the growth and elongation of the EC cell, and, in nhr-31 RNAi animals, excess lipid synthesis and other factors involved in cellular growth proceed unchecked leading to the production of new EC cellular material, even as this cell is no longer growing lengthwise. In this scenario, actively growing regions of the excretory cell could not expand laterally in either direction; consequently, excess cellular material would accumulate in varicosities that continue to grow larger even after animals reach adulthood. It is astonishing that NHR-31 controls such a small and specific set of target genes, and that nearly all of its targets comprise subunits or cofactors of the vATPase. While the fundamental conclusions of this study are not dependent upon the mechanism by which NHR-31 regulates gene transcription, NHR-31 is a transcription factor of the nuclear receptor type, and therefore it is tempting to propose that NHR-31 regulates the vATPases in response to a ligand signal by directly binding to the vATPase promoters. Consistent with this hypothesis, our binding site analyses of the vATPase promoters revealed a significant enrichment of nuclear receptor response elements in the form of ER6 or ER8 everted repeats with an AGTTCA consensus half site (Figure 6A and Table 4). The fact that this response element does not perfectly match the preferred response element architecture of the mammalian HNF4α is not surprising, as C. elegans contains dozens of HNF4-like receptors, and it is likely that NREs have evolved in nematodes in order to distinguish between NHR paralogs. We did, however, find strong enrichment of classical HNF4α binding sites (DR1 and DR2) in the promoters of the mouse vATPase genes, suggesting that coordinate regulation of the vATPase by HNF4 type receptors may be well conserved in mammals, even though the exact response element architecture may have changed. The physiological functions and target genes of nhr-31 have not been previously linked to an HNF4-type receptor, or any other nuclear receptor. NHR-31 shares a high degree of homology with mammalian HNF4 receptors, including nearly perfect conservation of key DNA binding elements and a strongly conserved ligand-binding domain (LBD). Interestingly, it has been proposed that the mammalian HNF4 receptors interact with free fatty acids and fatty acyl-CoA molecules [41], [42]. An ability of NHR-31 to bind to the acyl chain of a fatty acid or lipid molecule would provide a provocative explanation for how NHR-31 may be coordinating membrane synthesis and cellular elongation in the EC, which is likely to be occurring at sites distant from the nucleus. Because intensive membrane synthesis, transport, and fusion must take place in order to meet the needs of a growing excretory cell, such processes may release lipid based signals that activate or repress NHR-31 control of vacuolar ATPases and other genes associated with membrane biogenesis. Whether or not the functions of NHR-31 are conserved in mammals remains to be determined; however, the fact that both HNF4α and vacuolar ATPases are expressed at high levels in the proximal tubules of the mammalian kidney, combined with our demonstration that the mammalian vATPase genes contain a high density of HNF4α binding sites, implies that a functional role for HNF4 receptors in coordinate regulation of the vATPase in the renal system may indeed be a conserved process [16], [39]. The N2 Bristol strain of C. elegans was used for all experiments. Worms were maintained by standard techniques at 20–22°C. nhr-31 RNAi constructs were created by introducing the full-length NHR-31 cDNA into the L4440 RNAi feeding vector (Andy Fire, Stanford University). RNAi constructs for vha-5, vha-8, and vha-12 were obtained from the Ahringer RNAi library (University of Cambridge, Cambridge, UK). All RNAi constructs were transformed into the HT115 strain of E. coli and RNAi was introduced to N2 worms by RNAi feeding. RNAi expression was induced in the feeding bacteria by growing bacteria on NGM plates containing 3 mM IPTG and 100 µg/ml carbenicillin. Bacteria containing an empty L4440 RNAi vector were used for the RNAi control. Although NHR-31 is part of a large family of related nuclear receptors, these receptors have extensively diverged from one another during evolution, such that the closest paralog of NHR-31 shares only 55% homology in cDNA sequence; therefore, it is highly unlikely that there will be cross reactivity of the RNAi. Furthermore, C. elegans RNAi prediction programs do not indicate any cross reactivity (www. wormbase. org) [28]. Finally, the fact that nhr-31 (+/−) heterozygotes displayed similar EC defects further supports the specificity of this RNAi construct (Figure S1). An nhr-31 promoter/gfp reporter construct (Pnhr-31: : gfp) was generated by fusing ∼3 kb of upstream regulatory sequence and 17 base pairs of the first nhr-31 exon to the gfp gene, primers were created using the nhr-31a. 1 predicted isoform. Promoter DNA was amplified from genomic DNA using the following primers: NR-31UPGF (5′-TAA CTC GAG GAC GCA GGA AAG TCG GCA GTA GG-3′), as the 5′ upstream primer and NR-31-ExonI (5′-TCA CCC GGG TAC TCC CAA TCT TCG A-3′) as the 3′ downstream primer. Amplified DNA was inserted into the L3691 GFP reporter vector (from Andy Fire, Stanford University). The Pnhr-31: : gfp reporter vector was introduced into N2 worms by microinjection at a concentration of 50 ng/µl, worms were selected by EC fluorescence and no co-injection marker was used. Worms harboring the Pnhr-31: : gfp transgene were examined by both standard fluorescence microscopy and confocal microscopy. Images were taken using AxioVision 4. 6 software in multi-channel acquisition mode with an AxioCam MRU camera (Carl Zeiss Microimaging). For observation, larval or adult worms were mounted on glass slides with 2% agarose pads containing azide. Stack images of animals treated with nhr-31, vha-5, vha-8 and vha-12 RNAi were taken in both the FITC channel (488 nm) and DIC channels. To measure excretory cell diameter, control and nhr-31 RNAi animals expressing Pnhr-31: : gfp (5–6 animals) were analyzed by taking images which captured 50–100 µm of the proximal, middle, or distal regions of the posterior excretory cell tube. Diameter measurements were taken every 4–5 µm within the imaged regions using Zeiss measurement software. Data were plotted using Origen 5. 0 (OrigenLab, Northampton, MA) software, and data displayed in dot plots reflected values from each independent measurement, along with the mean, and standard deviation from the mean. Day 2 adults or L1 larvae were placed into a 20% BSA/PBS buffer solution and prepared in a Leica-Impact-2 high-pressure freezer according to the following protocol: 1) 60 hours in 100% acetone and uranyl acetate at −90°C. 2) Temperature was ramped from −90°C to −25°C over the course of 32. 5 hours. 3) Next, sample was incubated at −25°C for 13 hours. 4) Next, the temperature change was brought from −25°C to 27°C in a 13 hour temperature ramp. Serial sections were post-stained in uranyl acetate followed by lead citrate. Thin cross sections were taken from resin-embedded clusters of young adults or L1 larvae. Sections for nhr-31 RNAi and control RNAi adult animals were obtained from 5 different animals, and sections for L1 larvae were also taken from 5 independent animals. Synchronized L1 populations were prepared by hypochlorite bleaching of gravid N2 adults according to established protocols [6]. Synchronized L1 larvae were grown on control RNAi bacteria or nhr-31 RNAi until animals reached the early L4 stage of development. Worms were then harvested in M9, washed five times and immediately frozen in liquid nitrogen. RNA was extracted using a TRIZOL based method as described [6]. RNA was then labeled with Cy3 or Cy5 and hybridized to Washington University manufactured C. elegans microarrays (http: //genome. wustl. edu). Data were obtained from three independent biological replicates and analyzed using GenePix Pro 6. 0 software (Molecular Devices, Sunnyvale, CA). Ratios were calculated using background corrected, and normalized data (global mean). For QRT-PCR, RNA was extracted and cDNA was prepared using our previously published protocol [6] with the following exception: RNA was separated from genomic DNA with a Turbo DNA free prep kit from Ambion (Austin, TX). qPCR was performed using a BioRad iCycler (MyiQ Single Color, Bio-Rad Laboratories, Hercules, CA). The data were analyzed as previously described [6]. QRT-PCR primers amplified ∼100 base pair regions of NHR-31 target genes. Primers were designed using Primer3 software and calibrated by serial dilution of cDNA and genomic DNA. Primer sequences are available upon request. Worms treated with control RNAi or nhr-31 RNAi from the L1 to L4 stage of development were plated on high salt (500 mM NaCl) NGM-Lite plates seeded with E. coli. After various periods of high salt exposure, worms were scored for the ability to survive when rescued to a standard salt plate. Data for each time point was obtained from 250 animals. For rescue, worms were collected from the salt plates using M9 buffer+300 mM NaCl and transferred to standard NGM plates containing 50 mM NaCl. Worms were scored for survival after 12 hours of recovery [28]. To identify putative nuclear receptor response elements (NREs), we use the online computer program NHR-scan (http: //nhrscan. genereg. net), which was first presented in a study by Sandelin and Wasserman [34]. The promoters of C. elegans vATPase genes were defined as the sequence between the ATG translational start site of the vha gene of interest and the beginning or end of the next upstream gene in the C. elegans genome. For vATPase genes expressed in operons, the promoter was chosen using the ATG translational start site of the first gene in the operon. For mouse promoters, 2000 nucleotides of upstream sequence were extracted from each vATPase gene. This sequence included 1950 nucleotides upstream of the translational start site +50 nucleotides of coding sequence. In all cases, the isoform with the most 5′ translational start site was selected for promoter sequence extraction. To calculate and display the consensus half sites shown in Figure 6, all half site sequences were analyzed using the WebLogo online program (http: //weblogo. berkeley. edu/logo. cgi) [43]. | Title: The Caenorhabditis elegans HNF4α Homolog, NHR-31, Mediates Excretory Tube Growth and Function through Coordinate Regulation of the Vacuolar ATPase Summary: The function of many important biological structures requires the construction of very complex cellular shapes. For example, mammalian kidneys or related renal systems in other animals rely on the formation of elongated tubes that maximize surface area to facilitate the exchange of ions between the body and excreted fluid. Defects in kidney development or function may lead to kidney failure or polycystic kidney disease. Mechanisms involved in orchestrating the formation and function of the elaborate tube structures in renal systems are still poorly characterized. Here, we show a novel transcription factor involved in the growth and elongation of an excretory tube in C. elegans. This factor helps manage tube development by regulating genes involved in ion transport and membrane fusion, likely helping to balance the growth of the inner and outer portions of the excretory tube as this structure elongates. This transcription factor shares significant homology with a mammalian protein that participates in hormone signaling and is present in the kidney tubules, suggesting that elongation and growth of tube structures may rely on a new kind of hormonal communication that occurs between distant parts of the cell; this signaling mechanism may be important for appropriate kidney development in humans. | 10,428 | 294 | lay_plos | en |
Summarize: The girlfriend of one of Britain's biggest drug barons helped him run his international cocaine empire from her home in a picturesque town in the Cotswolds, a court heard. Anni Rowland worked as a personal assistant for convicted drug smuggler Kevin Hanley and arranged his travel, meetings and money transfers from her home, it is claimed. Hanley, 52, was jailed for 17 years in October after he brought cocaine into the country by hiding the drugs in shipments of watermelons, pomegranates and broccoli. Anni Rowland (left outside court today) worked as a PA for drug smuggler Kevin Hanley (right), who was jailed for 17 years last October after he brought cocaine into the country by hiding it in fruit, the court heard. Hanley's organisation broken up in 2012 after a National Crime Agency surveillance operation led to the arrest of his gang and the seizure of £2.5m of cocaine (pictured above) and £2m in cash at London property. Hanley's organisation broken up in 2012 after a National Crime Agency surveillance operation led to the arrest of his gang and the seizure of £2.5million of cocaine and £2million in cash. Police who raided the Chelsea home of Hanley's co-conspirator John Fowler also found £100,000 of amphetamines and £61,000 of skunk cannabis. Rowland was involved in the conspiracy with Greek television presenter Chrysi Minadaki, 45, who has also been jailed for her role in the conspiracy, the Old Bailey heard today. The 52-year-old, who is the mother of Hanley's child, commuted between her home in Stow-on-the-Wold, Gloucestershire, and London for her role in the conspiracy, the jury was told. She is on trial with Samson Karahassan, 59, who is said to be the manager of the Southall based fruit company Paris Weston, which was the front for the drug operation. Prosecutor Richard Jory said the drugs ring involved dozens of people across the world and was ‘well planned and well organised'. Police who raided the Chelsea home of Hanley's co-conspirator John Fowler also found £100,000 of amphetamines and £61,000 of skunk cannabis. Above, some of the £2million found at the property. Hanley's co-conspirators John Fowler, left, and Greek television presenter Chrysi Minadaki, right, who have been jailed for 17 and 16 years, respectively, for their roles in the conspiracy. ‘The drugs and cash may represent only a fraction of the overall drugs imported and the cash generated,’ he added. The cocaine began its journey in Venezuela before travelling to Greece where it was packed into loads of fruit and vegetables bound for the UK. Mr Jory said Rowland ‘was a personal assistant to Hanley who was the main organiser of this conspiracy'. She booked flights and accommodation for Hanley and Fowler around Europe. ‘She made charges for payment where money became due. She arranged for the transfer of money to Hanley and she occasionally attended meetings with him.’ The prosecutor added that Karahassan acted 'in a supervisory capacity'. 'His task was to sell the large amount of fruit and vegetables after it had been used as a cover load for the drugs,’ Mr Jory said. Rowland denies conspiracy to supply cocaine amphetamines, cannabis and money laundering. Karahassan, from London, denies conspiracy to supply the drugs. Fowler, 58, was earlier jailed for 17 years and Minadaki was sentenced to 16 years in prison. The trial continues. Sorry we are not currently accepting comments on this article | Summary: Anni Rowland, 52, was girlfriend of drug baron Kevin Hanley, court heard. She worked for him and arranged his affairs from their home, jury was told. Hanley, 52, is serving 17-year jail term for bringing cocaine into Britain. He hid drugs in fruit shipments in order to smuggle it into the country. Samson Karahassan, said to be owner of fruit company, is also on trial. | 870 | 100 | cnn_dailymail | en |
Write a title and summarize: The early stages of the thermal unfolding of apoflavodoxin have been determined by using atomistic multi microsecond-scale molecular dynamics (MD) simulations complemented with a variety of experimental techniques. Results strongly suggest that the intermediate is reached very early in the thermal unfolding process and that it has the properties of an “activated” form of the native state, where thermal fluctuations in the loops break loop-loop contacts. The unrestrained loops gain then kinetic energy corrupting short secondary structure elements without corrupting the core of the protein. The MD-derived ensembles agree with experimental observables and draw a picture of the intermediate state inconsistent with a well-defined structure and characteristic of a typical partially disordered protein. Our results allow us to speculate that proteins with a well packed core connected by long loops might behave as partially disordered proteins under native conditions, or alternatively behave as three state folders. Small details in the sequence, easily tunable by evolution, can yield to one or the other type of proteins. In addition to the folded and unfolded states, many proteins may adopt stable conformations that display mixed properties of the native and denatured states. These conformations, usually known as intermediates, may appear under unusual external conditions (i. e. non-physiological pH, pressure or temperature), in the presence of high concentrations of certain cosolutes (denaturants, salts), or as a consequence of mutations [1]–[9], and are supposed to be populated during folding (and unfolding), especially in the case of medium or large proteins [10]. Such folding intermediates may be on-pathway, facilitating the reaction, or off-pathway, acting as traps that may lead to missfolding and even aggregation [11]–[12]. The occurrence of equilibrium intermediates is often associated with stress phenomena and can trigger pathological effects, such as spongiform encephalopathy and other types of amyloidosis [13]–[14]. This explains the existence of many physiological mechanisms designed to reduce their harmful effects, mainly by reducing the life time of these potentially dangerous conformations [15]–[17]. For some proteins, however, physiological roles have been postulated for their intermediate conformations and such possibility might be more common than originally believed [8], [18]–[21]. All these reasons explain the interest in understanding the nature of intermediates and the atomistic details that favours the transition from native to intermediate structures. Unfortunately, the study of intermediate conformations is much more difficult than that of native forms. Equilibrium intermediates can be detected in vitro as a deviation from two-state behaviour, i. e., non-coincident protein unfolding curves obtained with different experimental techniques [22], although their structures and energetic properties are more difficult to probe. Folding intermediates are elusive to X-ray crystallography and their normally small population and the extensive signal broadening compared to the native state difficult their analysis by means of NMR techniques [23]–[28]. As a consequence, structural information on intermediates is often obtained by using low-resolution techniques, often based on Φ-analysis [29], [30]–[31], or low-resolution spectroscopic or scattering data, which can give clues on the general shape of the protein, but not atomistic information. This explains the need and frequent use of simulation techniques, particularly molecular dynamics (MD) to try to gain atomistic details that are unreachable to experimental techniques [32]–[44]. Flavodoxins are a family of proteins essential for the survival of many human pathogens that has become one of the most studied models for protein folding and unfolding. They are mono-domain α/β proteins, with a parallel five-stranded β-sheet surrounded by five α-helices, and they carry a non-covalently bound FMN group which can be reversibly removed [45]. Several experimental studies on apoflavodoxins from the Anabaena [46]–[47], Azotobacter and several Desulfovibrio [48]–[50] strains have demonstrated the thermal unfolding of this protein follows a three-state mechanism, where a partly unfolded intermediate accumulates at moderately high temperatures. Using a variety of techniques applied to wild type and mutant proteins Sancho' s group arrived to a low resolution picture of the thermal intermediate of the apoflavodoxin from Anabaena PCC 7119 [51]–[52] finding evidences that the intermediate is in fact close to the native structure, with the two hydrophobic cores well preserved, and with distortions probably located mostly in the loops and in one of the β-strands [53]. The overall dimensions of this thermal intermediate were characterized by small-angle X-ray scattering analysis, which suggests that the intermediate is slightly more extended than the native form, but clearly far from the expected situation of a random coil [54]. In this paper, we present a massive molecular dynamics (MD) effort for the study of the early stages of thermal unfolding of apoflavodoxin from Anabaena and for the characterization of its thermal intermediate. The study is especially challenging, since the slow folding dynamics of this protein (average transition times in the order of 101–102 millisecond makes impossible the use of pure force-approaches based on atomistic potentials, which would require second-scale trajectories. Furthermore, many attempts to use coarse-grained potentials failed to sample structures reproducing experimentally known intermediate properties and unfolding pathways, while equilibrium dynamics obtained from coarse-grained potential seems stiffer, but qualitatively similar to that expected from MD simulations (data not shown but available upon request). Accordingly, we decided to use a hybrid approach, based on the use of microsecond scale atomistic MD, supplemented by low- resolution spectroscopic and scattering data and previously derived Φ-analysis. The approach allowed us to characterize with atomistic detail the ensemble of conformations that define the intermediate as experimentally detected in melting experiments. With this synergistic approach the mechanism that drives the transition from native to intermediate and most likely the early stages of the thermal unfolding of the protein were explored. The crystal structure of Anabaena apoflavodoxin deposited in the Protein Data Bank with reference 1FTG [51] was used as starting conformation for our simulations. Crystallographic SO42− was conserved and simulated together with the protein [in the X-ray structure of Anabaena apoflavodoxin (crystallized in high ammonium sulphate concentration), a sulphate ion is bound, mimicking the FMN phosphate, which opens the possibility that the native conformation in this region is a consequence of the binding of the ion], the rest of ions 24 Na+ and 6 Cl− which are needed to neutralize large values of electrostatic potential around the protein were added using CMIP calculations implementing Poisson-Boltzman potentials [55]. The resulting systems were then solvated by around 7600 TIP3P water molecules [56], partially optimized, thermalized to 300 K (Nose-Hoover thermostat) and equilibrated using our standard protocol [57], followed by additional 50 ns of post-equilibration. Ten randomly selected snapshots (separated by at least 1 ns) were selected from the last 20 ns of the equilibration trajectory to generate the starting coordinates of ten replicas of the protein in water at T = 300 K. To increase diversity in the ensemble of the native form velocities were randomized and each replica was re-equilibrated for 5 ns prior to 0. 2 µs isothermic-isobaric production simulations (T = 300 K, P = 1 atm). The structure obtained at the end of the 50 ns equilibration at T = 300 K was heated slowly (0. 5 ns) to 368 K and equilibrated at this temperature for additional 10 ns, followed by 2 µs simulation using isothermic-isobaric conditions (T = 368 K, P = 1 atm). Periodic boundary conditions and Particle Mesh Ewald calculations were used to deal with long-range effects [58]. RESPA (Multiple time step) [59] with a minimum time step of 1 fs was used in conjunction with RATTLE [60] algorithms for maintaining bonds involving hydrogen atoms at equilibrium distances. Multi-microsecond trajectory at high temperature suggests that under the simulation conditions the unfolding trajectory reaches conformations which reproduce known properties of the thermal intermediate in less than 200 ns (see Results). Thus, to enrich our trajectories with the intermediate sate we performed 50 independent simulations starting from 50 different snapshots of the solvated protein extracted every nanosecond during the first 50 ns of the long T = 368 K simulation. Velocities in each snapshot were randomized and after 5 ns re-equilibration the 50 independent trajectories were followed for 0. 2 µs using identical simulation conditions, representing an aggregate time in the replicas of 12 µs. Such meta-trajectory was analyzed to determine the nature of the intermediate by confronting collected structures with experimental observables of the intermediate state. All MD simulations were carried out with NAMD 2. 6 [61]–[62] computer program using the CHARMM27 [63]–[64] force field using the MareNostrum supercomputer at the Barcelona Supercomputer Center. Snapshots were saved every picosecond and submitted to a large variety of analyses. Basic geometrical descriptors were determined using the ptraj module of AMBER9 [65]–[67], clustering was done in function of the RMSd of the clustered structures using the MMTSB Tool set [68] and representative structures of the clusters were determined as those closer to the centroid of each cluster. Secondary structure assignment and solvent accessibility of the representative structures of each cluster were calculated independently using the program PROCHECK [69]. Theoretical changes in the UV spectrum of the protein related to unfolding were determined by analysing the solvent accessible surface of the four Trp (SASTrp) and using four references: i) the crystal structure, ii) the ensemble obtained in MD simulations at room temperature, iii) four isolated Trp and iv) the protein after 50 ns of MD simulation at T = 500 K (where it reaches RMSd>15 Å from X-ray structure and all structural signatures are lost). SAS were computed using the NACCESS [70] program with standard values for protein and solvent particles. Essential dynamics (ED) [71] was done to determine the nature of the easiest deformation movements in the native and intermediate states of the protein and to determine the overlap between the essential deformation modes of the protein and the native<$>\raster=" rg1" <$>intermediate transition vectors. For this purpose covariance matrices were calculated for the native and intermediate ensembles (using a common reference system defined by the structurally conserved regions of the protein). Such covariance matrix was diagonalized to obtain a set of eigenvectors (the essential deformation modes) and the associated eigenvalues (the amount of variance associated to each eigenvector). The similarity between the essential space of native and intermediate was compared using Hess metrics [72]–[73] taking 50 eigenvectors as a common essential space (at least 90% of variance explained in each ensemble): (1) where n is the dimension of the essential space, A and B are two ensembles and stands for the eigenvectors. Considering the relative size of the protein and the essential space, any >0. 1 signals a statistically significant similarity [74]. The relative similarity between two essential deformation spaces was computed using [73]: (2) where the self-similarity indexes where obtained by comparing two different parts of the ensemble. Relative similarity index corrects absolute metrics by the intrinsic noise of MD simulations. A value close or even greater than 1 indicates that considering the noise of the trajectories the two ensembles are identical. The transition from the intermediate to the native states was obtained by taking the first eigenvector calculated by principal component analysis of a meta-ensemble obtained by mixing an equal number of snapshots of the intermediate and the native state. The overlap between the intermediate essential dynamics and the intermediate→native transition vector was determined as: (3) where Ov is the overlap (maximum equal to one), r is the transition vector and int stands here for the intermediate ensemble. Experimental φ-values profiles were taken from a previous work by Sancho' s group [75], [52], [53]. Theoretical estimates were derived by individual φicalc values (i stands for a residue) determined as the fraction of native contacts, Ni, made by that residue in the MD with respect to those found in the crystal structure, Ninat i. e., φicalc = Ni/Ninat [76]. Comparison between experimental and simulated φ values was extended to all residues with φi<1 except for residues in helix 3, where experimental uncertainties in the determinations were large [75]. The ability of a structural ensemble to satisfy the experimental Φ-value profile was studied by analyzing the sum (over all residues) of the difference between predicted and simulated Φ-values: (4) SAXS experiments were performed on the high brilliance beamline ID02 at the European Synchrotron Radiation Facility (ESRF, Grenoble, France). An apoflavodoxin sample at 1 mg/ml concentration was prepared in 50 mM Mops buffer at pH 7. Several SAXS curves were acquired with a momentum transfer range of 0. 07<s<0. 31 Å−1 at a broad range of temperatures (6–67°C). Solutions were pushed in a capillary into the chamber where they were equilibrated for five minutes. An equivalent protocol was applied to measure buffer profiles. Ten successive frames of 1 s each were acquired for both sample and buffer. Each frame was inspected and the presence of protein damage was discarded. The different scans at each temperature were averaged and subtracted from their buffer counterpart using standard protocols with PRIMUS [77]. The forward scattering, I (0), and the effective radius of gyration, Rg, was obtained from the scattering profiles using the Guinier' s approximation [78] assuming that, at very small angles (s<1. 3/Rg), the intensity can be represented as I (s) = I (0) exp (− (sRg) 2/3). SAXS curve measured at 26°C was used to evaluate MD trajectories in native conditions. The evaluation of trajectories in denaturing conditions was performed with the curve obtained from the Multivariate Curve Resolution by Alternating Least Squares (MCR-ALS) analysis of the SAXS dataset measured at the complete range of temperatures used to follow thermal denaturation of apoflavodoxin [54]. Principal Component Analysis (PCA) of the temperature variation SAXS dataset identified three components in the apoflavodoxin denaturation process that were assigned to the native the unfolded, and an intermediate states. MCR aims at finding the pure SAXS curves of these coexisting species in solution as well as the evolution of the relative concentration of these species upon environmental changes. The decomposition is obtained by solving the matrix equation (5) where D is the SAXS data matrix, C is the matrix describing the contributions of the N components, ST is the matrix describing the instrumental responses of these N components, and R accounts for the residuals of the fitting. Details of MCR-ALS approach and its application to SAXS data can be found in the original publications. [79]–[82]. Due to the post-processing nature of the SAXS profile of the intermediate, no experimental errors are associated to the derived intensities. A homogeneous 7% of error was assumed for each of the intensities of the curve. The agreement of SAXS profiles with three-dimensional structures of the MD trajectories was evaluated with CRYSOL [83] using default parameters. The χ-value of the fitting between experimental and theoretical curves is used as a measure of the quality of fitting (the smaller the χ-value, the better the agreement). Note that due to the de-convolution process and the use of a small homogeneous error in the intermediate, larger χ-values are expected in the fitting of the intermediate than to that of the native state. Near-UV absorbance spectra of apoflavodoxin [51] at different temperatures were recorded from 250 to 310 nm in a Chirascan spectropolarimeter (from Applied-Photophysics) using 30 µM protein solutions in 50 mM Mops, pH 7 in a 4 mm path-length cuvette. The absorbance spectra of native, intermediate and unfolded Anabaena apoflavodoxin were then determined by deconvolution of spectra recorded at different temperatures, using equation: (6) where the observed absorbance value at a given wavelength and temperature, Y (λ, T), is a linear combination of the values of the different states, Yi (λ, T) and of their populations, Xi (T) [84]. On the other hand, the populations are calculated at each temperature from the free energy values ΔG1 and ΔG2 previously obtained by global fitting to the sequential three-state model of unfolding curves recorded using absorbance, fluorescence and circular dichroism [53]. Ten independent 200 ns long MD simulations suggest that the equilibrium structure of the protein in solution is close to that found in the crystal, without any clear unfolding tendency (Figure 1). The RMSd of trajectory from the crystal structure are always below 3 Å for all replicas, and seems quite stable after the first 10–40 ns where protein relax from lattice contacts existing in the crystal structure (see Figure 1). The general shape of the protein and the structural core is fully maintained (see Tm-score plot in Figure 1) and the most significant deviation from crystal state is a small expansion of the protein as a result of the removal of lattice constraints, a behaviour very commonly found in massive MD simulations of the proteome [85]–[86], which is visible in a small (in average around 0. 5 Å see Figure 1) increase in radii of gyration (which happens already in the post-equilibration phase) as well as in an increase around 13% in the solvent accessible surface without changes in the polar/apolar SAS (see Figure 1 and Suppl. Figure S1). This slight increase in the size of the protein when liberated from lattice constraints is reflected in a small increase in the Trp accessibility, a parameter that correlates with the UV spectra [87] of the protein (see Suppl. Figure S2). However, all changes in size and shape of the protein upon transferring from crystal to solution are small. Not surprisingly then, the scattering properties computed from the 2 µs ensembles agree very well with the experimental SAXS curve, and also math the conformational preferences indicated by the X-ray structure (see Suppl. Figure S3). Contacts between residues are massively preserved (see Suppl. Figure S1) and the few native contacts which are transiently lost are typically replaced by alternative contacts with neighbouring residues (see Suppl. Figure S1). Both α helices and β sheet elements remain fixed at crystal values, while there is a conversion of a portion of residues in β turn into coil conformations (see Suppl. Figure S1), which is localized in the loop regions. In fact, analysis of B-factors (Figure 2) obtained in the 2 µs meta-trajectory reveals that the regions of larger flexibility are located around the loops of the protein. It is worth noting that most of these loops appear with large B-factors in the crystal structure. However, two loops which are flexible in the simulation (loop 90–100, contributing to binding the cofactor; and loop 120–135, characteristic of long-chain flavodoxins and involved in the binding of partner proteins) appear with low B-factors in the crystal. Analysis of different crystal structures of this protein in PDB (including 1FTG used here as starting conformation) reveals that in the crystal all these loops are directly or indirectly constrained by intermolecular packing contacts, which suggests that the largest mobility found in our simulations cannot be considered a simulation artefact (see Suppl. Figure S4). Cartesian cluster analysis (Figure 3) reveals that around 91% of the time trajectories are sampling the same conformational basin, which is very close to the crystal structure (RMSd to the crystal 1. 5–2. 5Å). The trajectory also populates two alternative basins (two clusters with population 4% each; RMSds to crystal 2. 0–2. 5Å) that only differ in the conformation of the long loop characteristic of the long-chain flavodoxin family (including β6 and β7; positions 120–135) and, at a minor extent, in the 90–100 loop (that connecting β4 and α4). Conformational changes in the loops yield to a marginal loss of native contacts in the region (see Figures 2 and 3), without further changes in the global structure. In summary, extended MD simulations demonstrate that selected force-field and simulation conditions are able to represent the folded form of the protein, which seems to be quite rigid except for local movements in the aforementioned loops. It is never clear what is the effective temperature in a classical MD simulation, since it is force-field dependent [88]–[89]. It is then almost impossible to define a simulation temperature as to guarantee that a finite time simulation will populate the experimentally characterized thermal intermediate. Thus, as described in Methods, we decided to locate (by comparison with experimental data) the intermediate as a transient conformational ensemble populated during unfolding at high temperature (below water boiling point). The increase in the temperature does not lead to complete protein unfolding in 2 µs (see Figures 4–5), something that could be expected only in very fast-folder proteins, typically small proteins with simple kinetic folding mechanisms. The maintenance of TM-score and the hydrophobic solvent accessible surface demonstrate that the protein core is preserved even until 2 µs of trajectory at high temperature. However, although the general fold is maintained, structural distortions from native structure are significant at the end of the simulation (as noted in the large RMSd) and affect key elements of α and β secondary structure (Figures 4–5). Major distortions are first located at the loops, as expected from native simulations (see above), but are later propagated to the neighbouring elements of secondary structure (see Figures 4–5). Thus, the large movements of loop 90–100 lead to distortions in neighbouring helix α4, which is shortened in 0. 2–0. 5 µs part of the trajectory and is almost completely lost at the end of it. Similarly, distortions in the long loop 120–135 produce early in the unfolding trajectory the disruption of small β-sheet elements β6, β7 and β5b and the shortening of terminal helix α5. Large movements of other smaller loops like 53–62 and 75–80 lead also to distortions of neighbouring secondary elements (for example helix α3), but this happens late in the trajectory and is less dramatic than those noted above. Clearly, our long simulation has not statistical power to describe the intermediate, but suggests a general picture where the perturbation in the loops corrupts in a first step short elements of secondary structure, which has no impact in global structure, but later the α-helices segments are compromised which should eventually yield to the complete unfolding of the protein in longer time scales. Cartesian cluster analysis reveals significant population (more than 100 ns) of 5 structural families along the 2 µs trajectories (Figure 5), which illustrates the increasing level of deformation gained along the simulation. It is tempting to assign the most populated family (cluster 4) as the putative intermediate, but as discussed above there is no guarantee that effective microscopic simulation temperature matches the experimental macroscopic temperature at which the intermediate is detected. Accordingly, we cannot be sure which family represents better the intermediate ensemble and we do not know at which time frame intermediate is populated during our MD unfolding simulations. Clearly, comparison with experimental observables can help to locate the intermediate in our ensemble. The UV spectra determined experimentally for the intermediate (see Methods) is very similar to that of the native state, without the blue shift in the spectra which is clear in the unfolded state (see Suppl. Figure S5). Thus, we can be quite sure that the exposure of Trp side chains has not changed much from native to intermediate state. Based on this criteria the intermediate is detected during the beginning of the simulation (around 0. 2 µs; Figure 6), while structures sampled at the second half of the trajectory yield too exposed Trp to justify experimental spectra. The SAXS spectra of the intermediate is well reproduced in the region 0. 1–0. 3 µs and later in the second half of the trajectory (as noted in χ values in Figure 6). Finally, the Φ–profile (see Methods) computed experimentally is well reproduced in the 0. 1–0. 2 µs region, while structures collected before are too “native-like” and those collected later have advanced too much in the unfolding pathway. In summary, comparison with experimental data strongly suggests that the intermediate is going to be closer to clusters 1–3 than to the most populated cluster 4 (see Figure 6), and that it is reached quite fast (around 0. 2 µs) during our unfolding simulation. Following the findings obtained from the analysis of the 2 µs trajectory, which suggested that native→intermediate transition happens early in the simulation, we performed 50 independent 0. 2 µs trajectories, which combined provide us a 10 µs ensemble enriched in the intermediate state. All the different trajectories advance towards protein denaturation (see Figure 7), with a range of velocities that show a normal distribution with unfolding velocities ranging from 0. 4 to 0. 8 nm RMSd/0. 2 µs. The lack of unusually slow or fast unfolding pathways [90] suggests the existence of a unique mechanism for the transition from folded to intermediate state under the selected simulation conditions, which is characterized by first a focalization of structural deformations in loops (Figure 7) and later a transfer of such perturbation to the surrounding elements of secondary structure (see Figure 8), matching the general unfolding trends found in the 2 µs trajectory. Cartesian clustering of the 10 µs meta-trajectory allowed us to detect six major “states (clusters) ”, four of them with populations above 5%. Not surprisingly, the most populated one (69% of meta-trajectory) is that describing a near-native conformation, which appears populated in the beginning of all the individual trajectories. As the unfolding progresses, partially unfolded conformations, characterized by distorted loop conformations and partial losses of neighbouring secondary structure become populated (Figure 8). Thus, in structures assigned to cluster 2 (10% meta-trajectory, populated in 55% trajectories) the large movements of the long loop (120–135) have led to the loss of short β strand elements β6 and β7. Ensembles represented by clusters 3 and 4 (12% and 7% meta-trajectory, populated in 65% and 45% individual trajectories) are characterized by an advance in the distortion produced by loop oscillations, either to the helix α4 (cluster 3) or the helix α3 (cluster 4). Finally, the minor clusters 5 and 6 represent much more distorted conformations, where a significant amount of secondary structure is lost and the departure from native basin is quite evident (Figure 8). Clusters 5 and 6 account for less than 1% of the entire meta-trajectory and are sampled only in two of the individual trajectories (one for each), which suggest that they do not fit the experimental requirements of the intermediate. It is very tempting to try to identify one of the above mentioned clusters with the thermal intermediate, but analysis of the individual trajectories show that in reality clusters 2–4 and part of structures assigned to cluster 1 interchange in a fast way and share many common characteristics, with a well conserved central core and largely distorted loop regions. The fast and large movement of such loops (and neighbouring secondary elements) generates a large dispersion in the structures when projected into the Cartesian space, which is reflected in the different assignment of structures to different clusters, when they share many key structural characteristics. It is also worth to note that structures which are within the same cluster can yield very different values of some experimental observables (see Figure 9), while structures very distant in terms of RMSd, and accordingly assigned to the different clusters can be indistinguishable in terms of experimental observables (see Figure 9). In summary, it seems that the intermediate cannot be represented as a small ensemble defined as a narrow basin centered in a well-defined structure, but as a wide ensemble of conformations that cover a wide range of Cartesian space, but that share a common conformational core. We interrogate our 10 µs ensemble to determine how many of these structures fulfill all the experimental requirements of the ensemble known experimentally for the thermal intermediate. Considering a loosely criteria (SASTrp between 100 and 300 Å2, fitting the SAXs curve with a χ below 1. 5 and fitting the Φ-value profile with absolute accumulated error below 2) almost 30% of the ensemble is annotated as intermediate. If we assume that experimental measurements for the intermediate are very accurate and use a much more restrictive criterion (SASTrp between 100 and 300 Å2, χ<1. 0 and Φ-error<1. 0) the intermediate ensemble is reduced to around 10% of the meta-trajectory. Such an ensemble is contributed by all individual trajectories and is proportionally enriched with structures assigned to clusters 2–4, with no contribution of clusters 5 and 6. When analyzed, the intermediate sampling shows a quite interesting picture of the structure that is transiently populated during thermal unfolding of the protein (Figures 10–11). The structure has enlarged with respect to the solution ensemble and hydrophobic solvent accessible surface has increased significantly, a fingerprint of a partially unfolded structure. A significant number of native contacts (defined as those present in the solution ensemble) are lost, especially those involving the protein loops, which have disappeared completely (Suppl. Figure S6). However, the structure maintains still many native inter-residue contacts, mostly located in the central core, where the amount of secondary structure has decreased, but is still quite significant (Figures 10–11). Clearly, analysis of the results demonstrates that the intermediate is not an alternative structure of the protein, but has to be represented as a wide ensemble (average RMSd between structures in the ensemble is around 0. 6 nm; Figure 11). Two broad regions can be easily recognized in the protein: the central core, where the native fold is well preserved and the loops (including the long loop hosting a small β-sheet encompassing strands 6 and 7), which adopt a canonical random coil confirmation (Figure 11). It is very interesting to realize that the large flexibility movements governing the essential dynamics in the intermediate ensemble are already a maximization of the intrinsic deformation pattern of the native state of the protein (absolute similarity (γ) = 0. 52; relative similarity (κ) = 0. 76, see eqs. 1 and 2), as it was already suggested by B-factor distributions (see Figure 2). Altogether, the intermediate fits perfectly in the definition of a partially disordered protein with a solid-like core and a liquid-like external loop core. It is very encouraging that such a representation of the intermediate fits well with the picture derived from the analysis of the NMR spectra of a mutant, which is believed to adopt intermediate-like conformation under native conditions [91]. Our MD simulations suggest a quite complete picture of the initial stages of the thermal unfolding of apoflavodoxin, which might be common to other proteins having long loops stabilized by weak contacts. Thus, under native conditions the protein has an intrinsic tendency to become a partially disordered protein, but several loop-loop contact keep the potentially flexible part of the protein reasonably organized. When the temperature increases these loops gain kinetic energy and in a quite short period of time become random coils (see Figure 2). The anchoring points of the loops, with the exception of short β-sheet elements, are very stable and held together the core of the structure defining the experimentally detected intermediate. Additional thermal energy will be then concentrated in the anchoring points of the loops, particularly in the helices 3 and 4, which are the Achilles' heel of the apoflavodoxin core. The distortion of these helices opens the structure and should lead to the final disruption of the three dimensional structure of the protein in longer time scales. Under this general picture, the lack of intermediate when denaturing agent is urea [92] can be easily rationalized, since urea will attack directly the core of the protein [39], eliminating the resistance points that stop the thermal unfolding pathways in a partially disordered conformation. Under native conditions the thermal intermediate acts as an “in-path” stationary state, since the essential deformations of the intermediate implicitly code the intermediate→native transition, as noted in the high overlap (Ov = 0. 63; see eq. 3) between the intermediate essential deformation subspace and the intermediate→native transition vector. This finding strongly suggests that the intermediate should be considered as an “activated-high entropy” form of the native state, (see RMSd oscillations in Figure 11), with properties of partially disordered protein, which acts as an attractor of folding routes toward a state that in the absence of an excess of kinetic energy will converge in a down-hill manner to the native form. We can hypothesize that a non-negligible number of partially disordered proteins, which adopt a well-defined three dimensional structure only in the presence of partner, can be considered as generalized examples of three-state folder proteins, which in native conditions populates conformations containing well-structured cores and very mobile regions. The flexibility pattern of such intermediates should favour a down-hill transition to a well-defined three dimensional structure in the presence of interactions stabilizing the disordered region (in these case binding partners). | Title: Defining the Nature of Thermal Intermediate in 3 State Folding Proteins: Apoflavodoxin, a Study Case Summary: A simplistic view of protein structure tends to emphasize the opposition between the native state and the denatured ensemble of unfolded conformations. In addition to these extreme conformations, proteins subjected to a variety of perturbations often populate alternative partly unfolded conformations, some of which are close in energy to the native state and, accordingly, can be populated under native or quasi-native conditions. There is increasing evidence that these "perturbed" conformations participate in protein function or, in some cases, are related to the outcome of folding diseases. We have used the "state of the art" molecular dynamics combined with a variety of experimental techniques to characterize for the first time, to our knowledge, the thermal intermediate of a three-state folding protein (apoflavodoxin). Based on our results we have been able to suggest a general mechanism of thermal unfolding in complex proteins and to determine interesting links between thermal intermediates and partially unfolded proteins. | 8,013 | 236 | lay_plos | en |
Write a title and summarize: By Emma JonesEntertainment reporter Speaking as the film had its world premiere at the Sundance festival in Utah, 26-year-old Radcliffe called making Swiss Army Man "one of the most joyous experiences of my entire life". Yet the media had differing opinions with Rolling Stone calling it '"Sundance's craziest movie" and the Guardian's headline reading "Daniel Radcliffe's flatulent corpse prompts Sundance walkouts" - a reference to the amount of people who deserted the premiere, apparently in disgust. From start to finish, Swiss Army Man is controversial. Paul Dano, currently starring in the BBC drama War and Peace, plays Hank, a lonely young man on the shore of a desert island. He is thinking about finishing it all, when the body of Radcliffe's character is washed up. Manny, as the corpse is called, can't control any of his bodily functions, but his gaseous presence saves Hank's life, and he's not prepared to let him go, taking him bodily back into civilization. Dano says he spent most of the weeks of filming "dragging Dan's corpse around the woods". But Radcliffe, far from having an easy job, says he found playing a dead body a difficult move. "It was a massive challenge physically," he says, "I mean he's dead, rigor mortis is setting in, so everything has to be said with the eyes. It was weirdly emotional, playing a corpse, but I'm really pleased about just how dead I look in the film." The actor, who after finishing Harry Potter, has taken parts such as beat poet Allen Ginsberg in Kill Your Darlings, and Igor in Paul McGuigan's gothic Victor Frankenstein, admits "a liking for the strange and fantastical". "Why did I take this part? Well, why not? I think it's a fantastic and important movie and it's just an amazing work of imagination." Kwan and Scheinert, Americans who met at a college animation class, collectively call themselves the "Daniels", and are known for making music videos, as well as a short film called My Best Friend's Sweating. They say that after writing Swiss Army Man they "thought we would just have to act in it ourselves, because the plot is so crazy, we really thought we would never get any actor to do it". Radcliffe says: "I didn't know what I was doing until I turned up, even though I had read the script. In fact I didn't know what I was doing from day to day. As you'll see if you watch the film, it was a hard one to be prepared for. But I had such a good experience. "A lot of my friends would say that playing a dead guy is a good role for me, I took some flak on that before I even filmed it. I don't want to say exactly what happens to me, apart from getting lugged around by Paul Dano, but his character uses and abuses my character's body. "It's going to split opinion, it's going to be divisive, and you're either going to love it or hate it. There's something very, very absurd about the movie." But Radcliffe denies that his heart now lies in independent film-making, saying "people should stop thinking big budget films aren't a challenge to make for actors". "I am sure I'll do one again sometime. For me, it's all about the freedom to do what project I want at the time. " Now living in New York, Radcliffe was due to take a part in a John Krokidas comedy about George W Bush's senior advisor Karl Rove, but the project is on hold. However, taking such a controversial role in Swiss Army Man will do his career no harm. According to trade magazine the Hollywood Reporter, it has all the makings of a cult classic. It says: "By turns enchanting, irritating, juvenile and yet oddly endearing… Swiss Army Man will probably make very little money theatrically. But over the long haul, there will be plenty of punters willing to watch it." Radcliffe himself says he has no regrets: "This is a film where cinematically, anything goes. It's crazy and wild. Am I happy I did it? You bet." The Sundance Film Festival runs until 31 January. Swiss Army Man is yet to receive a release date in the UK. | Title: Daniel Radcliffe's stiff Swiss Army challenge Summary: It's been the most talked about film at the Sundance Film Festival, and it stars Harry Potter actor Daniel Radcliffe. But Swiss Army Man, a feature film debut by directors Daniel Kwan and Daniel Scheinert, is achieving headlines for two reasons - not only has its sensationalist plot divided critical reaction, but Radcliffe plays a dead body with flatulence issues. | 987 | 96 | xlsum_en | en |
Write a title and summarize: Experimental studies have shown that some proteins exist in two alternative native-state conformations. It has been proposed that such bi-stable proteins can potentially function as evolutionary bridges at the interface between two neutral networks of protein sequences that fold uniquely into the two different native conformations. Under adaptive conflict scenarios, bi-stable proteins may be of particular advantage if they simultaneously provide two beneficial biological functions. However, computational models that simulate protein structure evolution do not yet recognize the importance of bi-stability. Here we use a biophysical model to analyze sequence space to identify bi-stable or multi-stable proteins with two or more equally stable native-state structures. The inclusion of such proteins enhances phenotype connectivity between neutral networks in sequence space. Consideration of the sequence space neighborhood of bridge proteins revealed that bi-stability decreases gradually with each mutation that takes the sequence further away from an exactly bi-stable protein. With relaxed selection pressures, we found that bi-stable proteins in our model are highly successful under simulated adaptive conflict. Inspired by these model predictions, we developed a method to identify real proteins in the PDB with bridge-like properties, and have verified a clear bi-stability gradient for a series of mutants studied by Alexander et al. (Proc Nat Acad Sci USA 2009,106: 21149–21154) that connect two sequences that fold uniquely into two different native structures via a bridge-like intermediate mutant sequence. Based on these findings, new testable predictions for future studies on protein bi-stability and evolution are discussed. New functional proteins are likely to evolve from existing proteins. Most existing proteins, however, are under selection to conserve their existing native structure in order to maintain functionality (and also to avoid aggregation and proteolysis). Without such selective constraints, the accumulation of random mutations would soon render a protein nonfunctional. When the same gene (protein) is under two selection pressures, i. e. to evolve a new functional structure while conserving its existing structure, an adaptive conflict arises. This adaptive conflict scenario is at the heart of most contemporary theories of molecular evolution, such as the popular Neofunctionalization and Subfunctionalization models (as reviewed in [1], [2]). However, these models generally require gene duplications to take place before adaptive conflicts can be resolved. This implies that such models can only explain long-term processes that involve many unlikely events, such as the occurrence of a suitable gene duplication event, followed by retention, fixation in the population, and additional beneficial or neutral point mutations in one or both gene copies. Only then would an adaptive advantage become possible. Because of these potential drawbacks, a more recent model (Escape from Adaptive Conflict, EAC) emphasizes the sufficiency of single-gene, multi-functional proteins during short term adaptive conflicts [3]. Similar ideas have been proposed earlier in terms of the concept of “gene sharing” [4], [5]. In fact, a gene duplication of a multi-functional protein is more likely to be successful than duplicating a protein with only a single function: first, because a new function is already present – thus it does not have to first evolve the new function in a rare mutant carrying a gene duplication; second, functional divergence can be faster because the multiple functions have already been responding to conflicting selection pressures; and, finally, retention and fixation of the duplication is more likely because the second copy can immediately provide higher activity levels through higher protein concentrations for the multiple protein functions, none of which would likely have been fully optimized in a single-gene, multi-functional protein. Indeed, there is increasing evidence that proteins have a significant capacity for multi-functionality. Not only are many enzymes known to exhibit promiscuity for nonnative reactions and substrates [6]–[8], multi-functionality has also been linked to proteins with two or more stable conformations [9]–[12]. These proteins can be called bi- or multi-stable. A few naturally occurring cases of such proteins are known, such as the prion protein that can assume different structures. One of these structures can aggregate to cause neurodegenerative pathologies such as mad-cow and Creutzfeldt-Jakob diseases [13], [14]. Protein bi-stability was also found in the cysteine-rich domain proteins (minicollagen) that form the walls of Cnidarian organelles called nematocysts [15]. Different conformers of these protein domains exhibit distinct patterns of disulfide bridges and perform different functions. Another example is the antiviral protein RhTC, which was found to target different HIV viruses by allowing a dynamic active site to assume very different conformations [16]. More generally, emerging evidence is lending support to the view that functional promiscuity in enzymes may frequently be based on thermodynamic fluctuations of conformational sub-states [17]. However, this may not always be the case, for example, if the functional promiscuity is mediated by changes of catalytic residues that do not cause conformational changes. An evolutionary theory of structure-based multi-functionality requires detailed knowledge of the sequence-structure relationship in proteins, as emphasized by the theory of neutral networks [18]–[23]. A neutral network consists of a connected set of sequences that fold into the same native (maximally stable) structure, and a pair of sequences in the network is connected if and only if they differ by one point mutation. Proteins can tolerate a number of mutations (mostly of surface amino acids [24], [25]) without losing their native structure. It has been shown experimentally that the neutral networks of two protein structures can be directly connected, such that one or two mutations can cause a switch from one native structure to the other [9], [26]–[28]. Because actual protein sequence space is too vast for computational — let alone experimental — exploration using current resources, we rely on a well-established explicit-chain biophysical model with exhaustive sequence-to-structure mapping [22], [23], [29]–[33] to provide a model of protein sequence space consisting of sequences with up to six-fold degenerate native state (i. e. proteins with up to six native structures). This model, termed the “hydrophobic-polar” (HP) model, is based on the central role of hydrophobic interactions in protein structures [29]. Earlier studies using the HP model but with non-degenerate native states have revealed that sequence space consists of distinct islands of neutral networks corresponding to unique native structures, which can be bridged either by single-site mutations (substitutions) [22], [32] or recombinational jumps [30]. A key feature of neutral networks arising from the HP model and similar simple exact models is a funnel-like distribution of free energy values around a most stable, and mutationally robust, prototype sequence [23], [34]–[36], or consensus sequence [37]. These funnels can act as attractors on evolving proteins outside the neutral network by allowing for selection of excited (non-native) conformational states, the stabilities of which increase with every incremental step toward the prototype sequence of that excited state [32]. More recently, the model was used to show an association between evolvability and phenotypic variation [33]. Some sequences in HP and HP-like models have been shown to have multiple native structure [23], [29] and even exhibit prion-like behaviors [38], [39]. However, an extensive account of sequence spaces with degenerate native structures is lacking and most theoretical studies of protein neutral networks to date have not considered the implications of multiple native structures [40]–[47]. In this context, our main aims here are to investigate: (i) where do bridge proteins preferably locate in sequence space, (ii) the manner in which bi-stability is distributed in the sequence-space neighborhood around bridge proteins, and (iii) the role of opposing selection pressures in the evolutionary dynamics that may take advantage of bi-stability. Toward these goals, we will first describe below the characteristics of the sequence space in our simple biophysical protein chain model. We will show that bridge proteins, and bi-stable proteins in general, have a high potential for facilitating evolution under adaptive conflicts. We will further demonstrate that this potential originates from a nonrandom distribution of bi-stability in sequence space. Subsequently, we will apply the concepts and insights gained from our simple model to real protein structures. In particular, we will describe bi-stability in a well-documented experimental case and also in a larger set of putative bi-stable proteins in the Protein Data Bank (PDB). Here we have employed a simple biophysical protein chain model to infer general properties of bi-stable proteins and their distribution in sequence space. The model used here is a simple exact model with an explicit representation of the protein conformations on a two-dimensional lattice. Despite their simplicity, such models capture essential features of the sequence-to-structure mapping of real proteins (see discussion in Results), and have provided significant insights into protein folding and evolution (reviewed in Refs. [31], [35]). The simple exact modeling approach allows a complete description of a system, but clearly such models are only a caricature of reality. In particular, only a limited variation of stability and bi-stability is allowed in our simple model, resulting in an appreciable percentage of sequences adopting two native structures with identical stability. Real proteins, in contrast, are unlikely to have exactly equal native stability in bridge proteins. Nonetheless, inasmuch as the goal of theoretical/conceptual models is to make predictions that can be tested experimentally, the main testable prediction of this work is that bi-stability can be increased or decreased by mutations leading either towards or away from bridge proteins, which are sequences that enjoy maximum bi-stability. While the fraction of actual bridge proteins is unknown, one may speculate how the HP model relates to real proteins. For example, consider the following argument: Our simple model only allows for 10 different energy states (HH contacts). If the conformational ensemble of a real protein was mapped onto 10 equally sized bins of energy, the lowest-free energy bin (highest stability) could contain two structures with similar yet non-identical stabilities such that the protein may function as a bi-stable bridge (e. g. see Table 2). This relaxed definition of a bridge could entail that one structure would still be much more stable than the other (as in the case of in Alexander et al. [27]). As a consequence, perhaps many such bridge proteins do not have easily measurable bi-stability because one structure remains dominant over the other. Nevertheless, the known examples of functional promiscuity suggest that even such unequal bi-stability may be of biological relevance. However, it is important to note that bi-stability can only occur if the two alternative native (or near-native) states are both thermodynamically accessible on time scales that are relevant for molecular functions. The consequence of bi-stability landscapes (Figure 2) for evolution is that proteins evolving under adaptive conflict for two alternative structures (whose extended neutral networks are connected in sequence space) are automatically directed towards bi-stable states, and that the dynamics of this process do not have to rely entirely on random genetic drift. Bridge proteins may thus be created in the laboratory by providing appropriate combinations of selection pressures, or known bridge proteins can be stabilized towards one of their structural sub-states. So far, this gradual shift in bi-stability was studied in terms of structural phenotypes; but the same concept should also apply to other definitions of phenotypes that depend upon structural stability. The simple fitness function in the present study rewards increased protein stability. This fitness function has provided significant insights; but it does not fully capture the subtle relationship between conformational stability and biological function in real proteins [82]. Too much stability can be detrimental for protein function, for example. More sophisticated biophysical models will need to be developed to incorporate such effects. Future work should also improve the computational methods for determining bi-stability changes of in-silico mutated PDB structures. In this regard, the discrepancy between FoldX and Rosetta predictions in Figure 3a is noteworthy. Using these algorithms, only local structural optimization around PDB structures for and was performed in the present study. We made no attempt in global structural optimization, which amounts to using an amino acid sequence as the only input to determine its native structure, i. e., solving the protein folding problem for the given sequence. For this much more challenging task, scoring functions such as Rosetta that rely on comparative modeling have difficulties when presented with sequences that have a high degree of identity but fold to different structures nonetheless. The ability of Rosetta to arrive at the correct structure can be greatly enhanced by considering not only the amino acid sequence but also including experimental NMR chemical shift data as input [83], as has been demonstrated for the system [84]. This finding underlines that the scoring function alone is insufficient for this system. As emphasized recently by van Gunsteren and coworkers, the energetics that govern the structural transition between and is highly delicate and cannot yet be accounted for atomistically using current force fields [85]. The quest for an accurate energy function for protein folding will likely remain a great challenge for years to come. In this light, the Rosetta criterion we adopted to obtain the present protein conformational diversity dataset (Dataset S1) is, inevitably, tentative. Nevertheless, based on the theoretical framework we developed and the general trend observed here, this dataset should serve as an impetus and provide useful candidates to be evaluated by future experimental investigations. Our evolutionary simulations (Figures 4, S3, and S4) are idealized scenarios that do not realistically capture evolution in natural populations, where usually only a small portion of sequence space would be explored by individuals within a population that are related to each other by common ancestry. Our master equation approach and the calculated steady state therefore only give a general evolutionary trend: given enough time and mutations, a population will acquire the most bi-stable proteins. Nevertheless, we have shown that the nature of bi-stability landscapes (Figure 2) – where incremental shifts of excited state stability can lead towards increased bi-stability – have the potential to speed up adaptation under adaptive conflict, whenever such stability shifts are advantageous. Evolutionary experiments will be needed to test these predictions under natural conditions. The increasing knowledge of promiscuous enzymes and the high evolvability of new enzyme functions [86] suggests that enzymes are in general mutationally robust for their native functions, while at the same time accepting mutations that enhance promiscuous functions. An apparently neutral mutation may therefore actually be adaptive. Even an apparently detrimental (destabilizing) mutation might promote a promiscuous function that is only beneficial under certain environmental conditions that the experimenter may not be aware of. The theory of neutral networks is impacted by the inclusion of degenerate native-state structures in that the notion of “neutrality” is moderated. While the strictest definition of neutrality (no change in protein activity/stability whatsoever) is usually not realistically applicable, a weaker definition (neutral, if the overall native structure is conserved, but a small loss of stability is tolerated) can be reconciled with experimental data. One can also go one step further and define neutral networks as fuzzy sets, where set membership is a continuous (not a binary) function over the interval [87]. Degenerate native states could be easily incorporated into such a definition. Our biophysical model shows that, at least in theory, excited state conformations may contribute to promiscuous functions, and could therefore be included into the “fuzzy” neutral set of all sequences that have some non-zero probability of forming that conformation. The membership to a fuzzy sequence set could be provided by the fractional population of the conformation (Equation 1 in Methods). Neutrality depends on the strength of the selection pressures involved, so that membership to a fuzzy neutral set as defined above requires a certain threshold of minimum stability. In the same manner as a falling sea level will expose more habitable land mass, a reduced selection pressure will allow for a larger number of viable protein variants. The intrinsic mutational robustness of neutral networks has been proposed to promote evolvability, i. e. the capacity to evolve towards new phenotypes [57], [88]. High robustness allows a population to accumulate many neutral variants within a neutral network. Some of these variants may be mutationally close to other phenotypes. We have shown that the inclusion of proteins with degenerate native states into neutral networks also enhances evolvability by providing more viable sequences between neutral networks. Compared to only proteins with non-degenerate native states, these additional sequences can access a substantial number of additional phenotypes. However, strong selection pressures would generally prevent evolution from utilizing degenerate native states, especially if only one of the native states is beneficial. The higher the native-state degeneracy, the lower the stability of a particular structure (see Figure 1b), and the lower the selection pressure would have to be for viability. If more than one native-state structure is beneficial, and if fitness effects are additive, a low stability may be compensated by providing multiple beneficial structures. Therefore, evolvability requires weak selection pressures in our model. Draghi et al. [88] have found an analytical solution to the general problem of how robustness and evolvability are related. Their results are general enough to be applied to any system (biological or non-biological) that exhibits robustness. In particular, they have provided a biological example of RNA phenotypes. However, their study does not provide any information specific to proteins, because the necessary parameters cannot be measured easily. Proteins are fundamentally different from RNA: structure formation in proteins is largely determined by hydrophobic-polar interactions, which are largely absent in RNA. Consequently, proteins and RNA do not share similar genotype-phenotype relationships [89]. The results from our simple protein model are consistent with the general predictions by Draghi et al. that evolvability increases with robustness, given two conditions: first, robustness is relatively low (only of mutations in sequences belonging to the same neutral network are neutral in our model; detailed data not shown); and second, only a small fraction of phenotype space can be accessed from each point in genotype space (true for our model, since the number of mutations per sequence is limited to 18, while phenotype space consists of 1475 stable structures [22]). One of the measures of evolvability that they use, and that we also have used here, is the number of mutationally accessible new phenotypes per genotype. An alternative measure is the time (e. g. number of generations) a population takes to adapt to a new beneficial phenotype. These two measures, however, capture different aspects of evolvability: one is the potential to quickly access many different phenotypes if the need arises (a concept followed by some experimentalists working on promiscuous enzymes [90]), while the other is adaptation to one specific phenotype that is under selection (a scenario we have investigated previously [32]). Here, we have followed the first approach of measuring evolvability, because we also impose the important additional requirement of conservation of the existing phenotype. With this restraint, the new beneficial phenotype is never fully reached by adaptation, especially since we refrain from a binary definition of neutral network membership (see previous section). Dual phenotypes (as exhibited by bi-stable proteins) have not been considered by Draghi et al. or any other theoretical study on neutral networks. By allowing dual phenotypes, which evidently also exist in nature, we allow an evolutionary compromise, whereas a binary definition of neutral networks completely prohibits adaptation as long as the need for conservation exists. In addition, our results also have consequences for the case of “unopposed” adaptation (without conservation), at least as far as modeling efforts are concerned: the true connectivity (evolvability) between neutral networks could be significantly underestimated, if proteins with degenerate native states are not considered. Both scenarios — a complete shift of selection pressures from one phenotype to another [32], [88] and adaptive conflict (present study) — are important fields of investigation since both are likely to exist in nature. The true robustness and evolvability parameters of proteins remain largely unknown. It appears plausible, however, that proteins may have become the dominant type of biopolymer (as opposed to RNA, or other unknown biopolymers that might have existed during early stages of evolution), in part because they produce the right balance between robustness and evolvability that allows for fast adaptation. Bi-stability as a factor for protein evolution (as opposed to conformational changes that are part of the same protein function) is currently based on a few mostly artificial example cases, but has not been widely observed in natural settings. This may be caused, in part, by experimental limitations in protein structure determination, and possibly also by a lack of research focus. Conformational diversity, as a more general case of bi-stability, has only recently gained broader attention [11], [59], [91], but much of its potential for evolution remains unexplored. We propose that bi-stability is particularly beneficial in complex and quickly changing environments that are likely to create adaptive conflicts. One important example could be the evolutionary arms-race between hosts and parasites. Bacteria and viruses have limited genetic material for adaptation to act upon, therefore these organisms might benefit from bi-stable and thus bi-functional proteins. Further studies in this direction will be instructive. Our model folds polymers of length that are configured on a two-dimensional square lattice. The model sequences have a binary residue alphabet (H for hydrophobic, P for polar). This simplicity makes it possible to enumerate all possible structures, or conformations (self-avoiding walks on the lattice) for all HP sequences. The energy function only includes one type of favorable energy, which is assigned for each hydrophobic intra-chain contact in any of the structures. Despite the simplicity in its construction, short-chain two-dimensional HP models have been shown to capture the essential physics of the sequence to native structure mapping of real proteins [29], [52]. The simplicity of the HP model allows for exact computation of the partition function — which takes full account of the energies of all structures, and thus permits an exact determination of the fractional population of each structure, which we use here as a stability measure. Specifically, gives the probability of a protein with sequence to fold into (adopt) structure: (1) where is the energy per hydrophobic-hydrophobic (HH) contact, is the number of such contacts in a conformation (thus total energy), is the Boltzmann constant and is absolute temperature. Conformation has HH contacts. The summation in Equation 1 is over all possible values in the entire conformational space, and denotes the density of states of sequence [23]. For any given HP sequence, the native-state degeneracy is the number of structures with the highest number of HH contacts,. In the present study, and were chosen to provide conditions generally favorable to the folding of sequences. As in some of our earlier studies [23], we have used throughout the present work. If the number of HH contacts in and are denoted by and, respectively, the difference for a given sequence is a measure of stability difference for that sequence (as used in Figure 2) because is directly related to the fractional populations and, viz., it follows from Eq. 1 that (2) The system of two adjacent neutral networks that we showed in Figures 2 and 4 as examples comprises one core neutral network (A; blue) with 48 sequences or the corresponding extended neutral network that includes an additional 84 sequences with, as well as another core network (B; red) with 20 sequences or the corresponding extended neutral network that includes an additional 40 sequences with. The Hamming distance between the two prototype sequences is 2, and the intra-chain contact difference between the native-state structures and is also 2 (Figure S2). In our model, the fitness of an HP sequence evolving under selection for two beneficial structures and is given by (3) where (4) and is an upper bound for the contribution of stability to fitness [81]. In all the computational results presented in this paper except those in Figure 5, the same was assumed for and for simplicity, whereas two different upper bounds and were used to gain a broader perspective in Figure 5. The upper bounds serve as a selection pressure because a low allows for destabilization of the protein, without fitness costs, whereas a high does not tolerate destabilization. Let be the set that contains all sequences in the extended neutral networks of two structures and. In our master-equation formulation of population dynamics [30], [81], the population of sequence at time is a function of sequence populations at time: (5) where and are, respectively, the mutation rate and sequence length chosen for the present study. is the population of at time, and is the population of the adjacent sequences of, denoted here by, in the sequence network, where two sequences are adjacent if and only if they can be converted to each other by a single mutation. The factor is introduced to keep the total population normalized to 1 to facilitate comparisons of distributions at different time steps. The factor is a reproduction term that is determined by the relative fitness of sequence, being the average fitness (weighted by population) of all at time. Population dynamics were calculated from an initial state () in which only the prototype sequence of one network (A) was populated (Figure S4). The steady state was reached by iterating Eq. 5 until the values remained essentially unchanged over many generations. For a given network topology, the steady state is independent of the initial state. In this regard, it should be noted that for some of the control calculations in Figure S3 only the initially populated network were populated at steady state because in those cases the two networks were disconnected by the artificial removal of bridge sequences in the control simulations. The above master-equation approach presupposes an effectively infinite population. To assess the effect of finite population on steady-state distributions, we have also conducted Monte Carlo simulations under the same general conditions with respect to selection pressure and mutation rate (Figure S4) [81]. Similar to the initial conditions in the master-equation formulation, every Monte Carlo simulation was initialized with a population consisting of identical individuals each carrying the prototype sequence. At each subsequent time step, a random number between 0 and 1 was drawn for each of the 18 monomers in each of the 1000 sequences. If the random number was less than, the monomer was mutated (or, depending on whether the initial monomer was H or P), and fitness was then recalculated in accordance with Equation 3. Multiple mutations in one sequence can occur in one time step; but these events were very rare under the chosen value for. Evolution thus proceeded essentially in discrete steps of single point mutations. After all mutations were performed for a given time step, a new population was selected for the next time step by the following consideration: As in the master-equation formulation, the relative fitness of individual in the population with fitness is, where. Let and for (thus). The' s resulting from this construction are the boundaries of discrete bins in with widths equal to the values. Now, to select an individual, a random number was drawn and individual was selected if. This procedure picks an individual by letting the random number fall into one of the bins. By repeating this procedure times, a new population of 1,000 individuals was selected. Because the same individual could be picked multiple times and some individuals might not be picked at all, fitter individuals would be statistically over-represented in the next generation, as they should. For illustrative purposes, the sequences belonging to the two adjacent neutral networks in Figure 2a were depicted as nodes placed by the Fruchterman-Reingold algorithm [92] that simulates physical spring forces between connected nodes. This algorithm serves to keep edge lengths as equal as possible, resulting in a network layout that roughly reflects the sequence connectivity relationships, i. e. sequences differing by many mutations are also farther apart in the two-dimensional node layout. Stability difference (see above) was then added as a third axis for the drawing in Figure 2a. The NMR model 1 of (PDB code 2FS1) and the X-ray structure of (PDB code 1PGA) were used as the wildtype structures in our analysis. The two wildtype sequences have a sequence identity of around. In addition to the wildtype pair, we considered also the sequence pairs in Refs. [27], [69] that are intermediate mutants between the two wildtypes and have pairwise sequence identity of,,,,, and. For any one of these sequences, only one — but not both — of the and structures was experimentally inferred to be native [27], the other was a hypothetical excited-state structure. To estimate the stability difference between excited- and native-state structures, we modeled the free energy of every sequence in both the and structures by “threading” each mutant sequence into a modified and a modified structure that were locally optimized for the given sequence. Two different methods, namely Rosetta and FoldX, were employed for this computation. In the Rosetta approach (PyRosetta v2. 0 implementation [93]), mutations were introduced using the “PackRotamersMover” routine to produce the sequence variants, then each of the two wildtype PDB structures embodying the mutant sequence were optimized using the FastRelax method, which is currently the best-performing free energy minimization method of Rosetta [71]. FastRelax was applied three times in a row to each wildtype PDB structure to ensure that the resulting structures were as optimized as possible and had comparable free energy scores. The same FastRelax procedure was also applied to the two wildtype sequences. Free energy scores were computed by the standard energy function of Rosetta with undamped Lennard-Jones repulsions (“hard rep”) [72]. In the FoldX approach, the mutagenesis engine (“BuildModel”) and the standard energy function of FoldX were used to generate and evaluate the sequence variants. For each sequence, the “Repair” function of FoldX was used to optimize the side-chain orientations. In contrast to the Rosetta approach that allows for movement of all atoms to achieve local optimization of the structure, FoldX (version 3. 0) [70] only optimizes side-chain orientations but leaves the backbone unchanged, resulting in less structural optimization (from the PDB wildtype) for any given sequence. A comparison of the performance of Rosetta and FoldX in our analysis of the system is provided in Figure 3c. To determine the hydrophobic contact density for a given all-atom protein structure (Figure 3d), the number of C-atom pairs from different amino acid residues and the total number of inter-residue atomic contacts were counted. An atomic contact is defined by an inter-atomic distance of less than. Computation of contacts was performed using the P3D Python module [94]. Among several possible choices of threshold separation, we found that a threshold separation of in the definition of produced the best illustration of the native-structure switch between and (Figure 3d). As in the lattice HP protein model [73], only contacts between residue pairs that are at least 3 positions apart along the chain sequence were counted in the measure. Conceptually, the difference in hydrophobic contact density plotted in Figure 3d for the all-atom protein structures corresponds roughly to, where and are, respectively, the total number of contacts of structures and in our biophysical protein chain model. We note that is a simple measure of hydrophobic contact density that does not rely on a hydrophobicity scale (e. g., that of Kyte-Doolittle [95]). It takes contributions from the carbon atoms in hydrophobic as well as non-hydrophobic residues. For instance, in the present application to the system, contains contribution from the C-atoms in the polar residue lysine in the core of both and [27]. All 7989 redundant protein structure clusters were obtained from the protein conformational database PCDB (version 2, August 2011) [59]. Each entry in PCDB contains a cluster of CATH [64] domain structures that correspond to the same sequence. The largest conformational difference (max PCD) between two structures of the same cluster was determined, using the RMSD values (in) that were already included in PCDB (obtained using MAMMOTH [59], [96]). Stability value of each structure in a pair with max PCD was calculated with Rosetta [93] by the standard energy function (see above). If a structure had unfavorable energy (), the FastRelax method (see above) was applied until a favorable energy () was reached. Potential bridge proteins were identified by the criteria described above in Results. The set of proteins we thus obtained is listed in Dataset S1 with the cause (s) of conformational diversity provided by PCDB. | Title: Evolutionary Dynamics on Protein Bi-stability Landscapes can Potentially Resolve Adaptive Conflicts Summary: Proteins are essential molecules for performing a majority of functions in all biological systems. These functions often depend on the three-dimensional structures of proteins. Here, we investigate a fundamental question in molecular evolution: how can proteins acquire new advantageous structures via mutations while not sacrificing their existing structures that are still needed? Some authors have suggested that the same protein may adopt two or more alternative structures, switch between them and thus perform different functions with each of the alternative structures. Intuitively, such a protein could provide an evolutionary compromise between conflicting demands for existing and new protein structures. Yet no theoretical study has systematically tackled the biophysical basis of such compromises during evolutionary processes. Here we devise a model of evolution that specifically recognizes protein molecules that can exist in several different stable structures. Our model demonstrates that proteins can indeed utilize multiple structures to satisfy conflicting evolutionary requirements. In light of these results, we identify data from known protein structures that are consistent with our predictions and suggest novel directions for future investigation. | 7,436 | 235 | lay_plos | en |
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Consistent with applicable law, we do not discriminate on the basis of race, creed, color, national origin, sexual orientation, lawful source of income, military status, sex, gender identity, age, disability, familial status (having children under age 18), or religion. Equal Housing Opportunity. This Hamptons property is one fisherman’s catch of a lifetime. An Amagansett South site that’s home to a rinky-dink two-bedroom trailer is on the market for $1.1 million. That’ll be a whale of a profit for owner Richard Lester, who told The Post he paid just $300 for the property in 1956 and added the $15,000 wood-paneled trailer a few years later. “I had to work my ass off to get that,” said Lester, 79. Lester still lives with his wife Tess in his modest 700-square-foot castle, which sits on 0.36 acres of land in the exclusive — and expensive — enclave, just a mile away from ritzy Indian Wells Beach. A nearby home recently sold for $4 million, and neighbors include Jerry Seinfeld and Paul McCartney. Lester, who still makes his living from the sea, said he never dreamed he’d land the big one — on land. “Not in God’s world,” he said. “I think it’s ridiculous!” Realtors said the price is all about the land and not the trailer, which looks like it could be straight out of “Deliverance.” “Erase the trailer from your mind and it’s $1 million for land,” said listings agent Ray Lord, from Douglas Elliman Real Estate. All potential buyers are builders, who plan to tear down the trailer and build a Hamptons dream home, Lord said. “No one, I imagine, would build less than a three-bedroom, three-bathroom house,” said Lord. And a shed on the property will likely be turned into a pool house. But the real-estate listing suggests homeowners could still live in the trailer while building their future mansion. “Use as is now, while you plan for your 4,000 square foot new house with 20 foot x 50 foot swimming pool,” it says. “We had one buyer that was a husband and wife. The husband suggested it and the wife was not keen on the idea,” said Lord. Run-down trailer in The Hamptons listed for $1.1 million By Katherine Biek Video transcript provided by Newsy.com There's only one mantra in real estate that really matters: location, location, location. This is why a run-down trailer in the Hamptons is on the market for $1.1 million. The 400-square-foot space boasts one bedroom and one bathroom. The property wasn't always this expensive. The New York Post talked to Richard Lester, the current owner. He said he originally bought the land — which is less than half an acre — in 1956 for $300. Talk about inflation. But the $1.1 million price tag sort of makes sense considering the prices nearby homes have sold for. Like the five-bedroom house that sold for just under $4 million in the summer of 2014. Celebrities like Jerry Seinfeld and Martha Stewart also have homes in the area. And the trailer's price is actually lower than the average listing price in the Hamptons, which is now around $1.7 million, according to CNBC. If trailers really aren't your thing, though, just do as the listing says and "Use it as it is now, while you plan for your 4,000 square foot new house with [a] 20 foot x 50 foot swimming pool." This video includes images from Getty Images. | Summary: In 1956, fisherman Richard Lester paid $300 for 0.36 acres of property in the Hamptons. Later, he added a $15,000 trailer. Now, the whole thing can be yours for the low, low price of just $1.1 million, the New York Post reports. "You are not dreaming," the listing reads at elliman.com, noting that a nearby house sold for $4 million. The situation, in Amagansett South, offers the unusual opportunity to simultaneously live in a trailer and have celebrity neighbors-in this case, the likes of Paul McCartney, Jerry Seinfeld, and, according to WSOC, Martha Stewart. The trailer, per the Post, is 700 square feet and "looks like it could be straight out of Deliverance." But a listings agent says you should ignore it: "Erase the trailer from your mind and it's $1 million for land," he says. "No one, I imagine, would build less than a three-bedroom, three-bathroom house" on the property, which is about a mile from Indian Wells Beach. The general location does seem to be a winner: Last year saw an East Hampton home sell for $147 million. | 1,028 | 276 | multi_news | en |
Summarize: CLOSE A man convicted of stalking Sandra Bullock has killed himself after a police standoff. Time Sandra Bullock in October 2015 in Los Angeles. (Photo: Richard Shotwell, Richard Shotwell/Invision/AP) Joshua James Corbett, the Sandra Bullock stalker convicted of breaking into her house in 2014, killed himself Wednesday in his Los Angeles County home after an hours-long standoff with police. The county coroner's office confirmed the identity of Corbett, 42, and that he was found dead in his La Crescenta home. Officer Rosario Herrera, a spokeswoman for the Los Angeles Police Department, said officers went to the home before 7 a.m. P.T. on Wednesday to serve a search warrant, linked in some way to the Bullock stalking case. The man in the home barricaded himself inside and refused to come out. After "threats of violence" were made at the scene, a SWAT team arrived. After five hours, police entered the house and found the man dead of "self-inflicted injuries" from a "sharp object," Herrera said. The coroner's office has not yet released details on the cause of death. The first page of a letter Joshua James Corbett wrote to actress Sandra Bullock, included as evidence in court case against Corbett for stalking the Oscar-winning actress. (Photo: AP) No shots were fired at any time, no one else was injured, and no one else was in the house, Herrera said. She said the officers who tried to serve the search warrant were seeking to arrest Corbett and that the intended arrest was linked to the Bullock case. Labeled an "obsessed fan," Corbett was convicted in 2017 of breaking into Bullock's home in 2014, forcing the Oscar-winning actress to hide in her closet while calling police. He was arrested inside her house when police arrived. Authorities later uncovered a cache of illegal weapons at his home, but all weapons charges were later dropped. They also found a two-page handwritten letter to Bullock from Corbett, who wrote: "You could of (sic) had me today however you choose other people over me. I'll be around as you know. I love you." Corbett was sentenced to continued mental health treatment and probation after pleading no contest to felony stalking and burglary charges. Corbett also was ordered to stay away from the actress and not attempt to contact her for 10 years. Read or Share this story: https://usat.ly/2rkWCmS FILE - In this March 4, 2018 file photo, Sandra Bullock arrives at the Oscars in Los Angeles. A man who killed himself during a standoff with Los Angeles police was convicted last year of breaking into... (Associated Press) FILE - In this March 4, 2018 file photo, Sandra Bullock arrives at the Oscars in Los Angeles. A man who killed himself during a standoff with Los Angeles police was convicted last year of breaking into Sandra Bullock's home and stalking the Oscar-winning actress, his lawyer said Thursday. Joshua James... (Associated Press) FILE - In this March 4, 2018 file photo, Sandra Bullock arrives at the Oscars in Los Angeles. A man who killed himself during a standoff with Los Angeles police was convicted last year of breaking into Sandra Bullock's home and stalking the Oscar-winning actress, his lawyer said Thursday. Joshua James... (Associated Press) FILE - In this March 4, 2018 file photo, Sandra Bullock arrives at the Oscars in Los Angeles. A man who killed himself during a standoff with Los Angeles police was convicted last year of breaking into... (Associated Press) LOS ANGELES (AP) — A man who killed himself during a standoff with Los Angeles police was convicted last year of breaking into Sandra Bullock's home and stalking the Oscar-winning actress, his lawyer said Thursday. Joshua James Corbett missed a court date last month and on Wednesday barricaded himself inside his house when police arrived to serve an arrest warrant, attorney Steve Sitkoff told The Associated Press. Corbett was scheduled to appear for a progress report as part of his probation in the Bullock stalking. SWAT officers were called after Corbett threatened to kill police, LAPD Detective Meghan Aguilar said. Corbett, 42, was found dead about five hours later at the home in the La Crescenta neighborhood. The death was reported as a suicide and an autopsy was planned Thursday, said coroner spokesman Ed Winter. Corbett's father had reported that his son, who suffered from mental health problems, hadn't been doing well in recent weeks, Sitkoff said. "He was due for a progress report and he just didn't want to go to court," the attorney said. "It's a very sad situation." Corbett pleaded no contest last year to felony stalking and burglary charges after being arrested inside Bullock's West Los Angeles home in 2014. He was sentenced to continuing mental health treatment and probation. He was also ordered to stay away from the "Miss Congeniality" actress and not attempt to contact her for 10 years. Bullock never personally appeared during the case, but her frantic 15-minute 911 call while hiding inside a closet was a key piece of evidence that led a judge to order Corbett to stand trial. Corbett received mental health evaluations while in custody and his attorneys had hoped to resolve the case with an agreement that ensured he received continued mental health treatment. He lurked outside the gates of Bullock's home for several days before hopping the fence on June 8, 2014, according to court testimony. He rang Bullock's doorbell for several minutes before entering her home through a sunroom door. The actress caught a glimpse of him as he walked past her bedroom door, allowing her to lock herself in a closet and call police. Corbett was unarmed but he had 25 pages of writings describing his obsession with the "Gravity" star and describing himself as her husband. Authorities later uncovered a cache of illegal guns at his home, but all weapons charges were dropped. ___ Follow Weber at https://twitter.com/WeberCM | Summary: The chilling story of Sandra Bullock's convicted stalker has come to a dramatic close. Joshua James Corbett, who was convicted of breaking into Bullock's home in 2014, killed himself Wednesday in his La Crescenta, Calif., home during a five-hour standoff with police, USA Today reports. Corbett, 42, barricaded himself in his home around 7am after officers arrived with an arrest warrant related to the Bullock case. A SWAT team was called in after "threats of violence," according to a police spokesperson. When the police eventually stormed the house, they found Corbett dead of "self-inflicted injuries" from a "sharp object," the spokesperson says. Corbett's conviction came after he broke into the Oscar-winning actress' home as she hid in a closet. He was inside her house when police arrived. After that incident, police found a cache of weapons in his home. Corbett's lawyer tells the AP the warrant was being served because Corbett missed a court date last month for a progress report hearing related to his probation in the Bullock case. "He was due for a progress report and he just didn't want to go to court," the attorney says. "It's a very sad situation." | 1,412 | 281 | multi_news | en |
Summarize: This invention relates to a process for the production of alkoxylated nonionic surtactants in which compounds containing active hydrogen atoms or carboxylic acid esters are reacted with alkylene oxides in the presence of optionally modified hydrotalcite as catalyst and optionally other selected co-catalysts and the reaction products obtained are aftertreated with acids. An important group of nonionic surfactants are products of the addition of alkylene oxides, especially ethylene oxide and/or propylene oxide, onto compounds containing active hydrogen which are normally produced by homogeneous catalysis in the presence of alkali metal hydroxides or alkali metal alcoholates. Products with a broad homolog distribution are obtained by the homogeneously catalyzed process. Products with a narrow homolog distribution can be obtained by carrying out the reaction in the presence of optionally modified hydrotalcites, for example in accordance with DE-A-38 33 076. The alkoxylation of carboxylic acid esters also takes place with better results in the presence of hydrotalcites, the alkylene oxides being inserted into the carbonyl ester bond, for example in accordance with the two patents EP-B1-0 339 425 and EP-B1-0 523 089. However, after the actual alkoxylation using optionally modified hydrotalcite, separation of the catalyst from the reaction product presents technical difficulties because the optionally modified hydrotalcite is generally so finely particulate that it can only be filtered through special filter candles. Unfortunately, the catalyst cannot be allowed to remain in the end reaction product either because otherwise clouding and sedimentation can occur. Accordingly, the problem addressed by the present invention was to provide a process for the production of alkoxylated nonionic surfactants which would not have any of the disadvantages of the complex filtration or clouding and sedimentation of the end reaction product. BRIEF SUMMARY OF THE INVENTION Surprisingly, the problem stated above has been solved by decomposing the catalyst hydrotalcite and any co-catalysts present by addition of acids after the alkoxylation rather than removing them by filtration. The invention includes the observation that the acid aftertreatment decomposes the hydrotalcite and any co-catalysts present into products which can remain in the reaction mixture without any clouding or sedimentation subsequently occurring. Accordingly, the present invention relates to a process for the production of alkoxylated nonionic surfactants by reaction of compounds containing active hydrogen atoms or carboxylic acid esters with alkylene oxides in the presence of optionally modified hydrotalcite as catalyst and optionally co-catalysts, characterized in that at least equimolar quantities—based on hydrotalcite and optionally co-catalysts—of acids are added after the alkoxylation. DETAILED DESCRIPTION OF THE INVENTION The compounds containing active hydrogen atoms may be selected, for example, from the following classes of compounds: a1) alcohols containing 6 to 22 carbon atoms (so-called fatty alcohols) such as, for example, caproic alcohol, caprylic alcohol, capric alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, palmitoleyl alcohol, stearyl alcohol, isostearyl, alcohol, oleyl alcohol, elaidyl alcohol, petroselinyl alcohol, linolyl alcohol, linolenyl alcohol, ricinolyl alcohol, elaeostearyl alcohol, arachyl alcohol, gadoleyl alcohol, behenyl alcohol, erucyl alcohol and the technical mixtures thereof obtained, for example, in the hydrogenation of methyl ester fractions of native origin or aldehydes from Roelen's oxo synthesis. Fatty alcohols containing 12 to 18 carbon atoms, for example technical coconut or tallow fatty alcohol cuts, are preferred. Another group of suitable fatty alcohols are the co-called Guerbet alcohols which are produced by the alkali-catalyzed condensation of 2 moles of fatty alcohol and which may contain 12 to 36 carbon atoms. a2) Carboxylic acids containing 6 to 22 carbon atoms (so-called fatty acids) and hydroxyfatty acids such as, for example, caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroselic acid, linoleic acid, linolenic acid, ricinoleic acid, elaeostearic acid, arachic acid, gadoleic acid, behenic acid and erucic acid and the technical mixtures thereof obtained, for example, in the pressure hydrolysis of natural fats and oils. Fatty acids containing 12 to 18 carbon atoms, for example technical coconut oil or tallow fatty acids, are preferred. a3) Alkyl phenols, polyglycols, fatty amines, vicinal hydroxy/alkoxy-substituted alkanes obtainable, for example, by ring opening of epoxide compounds with alcohols or carboxylic acids and secondary alcohols. Within the group of compounds containing active hydrogen atoms, the alcohols or carboxylic acids containing 6 to 22 carbon atoms are preferred. In another embodiment of the process according to the invention, carboxylic acid esters are used as starting materials. Basically, there are again two types of carboxylic acid esters, namely: b1) carboxylic acid lower alkyl esters corresponding to formula (I): R 1 CO—OR 2 (I) in which R 1 CO is an aliphatic acyl group containing 6 to 22 carbon atoms and R 2 is a linear or branched alkyl group containing 1 to 4 carbon atoms. Typical examples are esters of caproic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, palmitoleic acid, stearic acid, isostearic acid, oleic acid, elaidic acid, petroselic acid, linoleic acid, linolenic acid, ricinoleic acid, elaeostearic acid, arachic acid, gadoleic acid, behenic acid and erucic acid and technical mixtures thereof with methanol, ethanol, propanol or butanol. Methyl esters of fatty acids containing 12 to 18 carbon atoms and, more particularly, technical coconut oil or tallow fatty acid methyl esters are preferably used. b2) carboxylic acid glycerol esters corresponding to formula (II): in which R 3 CO is an aliphatic acyl group containing 6 to 22 carbon atoms and R 4 and R 5 independently of one another represent hydrogen or likewise an aliphatic acyl group containing 6 to 22 carbon atoms. Typical examples of such compounds are synthetic but preferably natural triglycerides, such as palm oil, palm kernel oil, coconut oil, rapeseed oil, olive oil, sunflower oil, cottonseed oil, peanut oil, linseed oil, lard oil, bovine tallow and lard. Castor oil or hydrogenated castor oil is preferably used. In one preferred embodiment, the full esters are replaced by fatty acid partial glycerides, more particularly monoglycerides of fatty acids containing 12 to 18 carbon atoms. Technical coconut oil fatty acid monoglycerides are particularly preferred. Within the group of carboxylic acid esters, carboxylic acid lower alkyl esters, especially the methyl esters of carboxylic acids containing 6 to 22 carbon atoms, are preferred. Optionally modified hydrotalcites are used as alkoxylation catalysts in the process according to the invention either on their own or in admixture with selected co-catalysts. In one embodiment of the present invention, optionally modified hydrotalcite is used on its own as catalyst. Calcined or hydrophobicized hydrotalcites, as known for example from German patent applications DE-A1 38 43 713 and DE-A1 40 10 606 (Henkel), are used as modified hydrotalcites. Calcined hydrotalcites are particularly preferred. In another embodiment of the invention, optionally modified hydrotalcites and selected co-catalysts are used together as alkoxylation catalysts. Suitable co-catalysts are compounds from the group consisting of hydroxides, oxides and/or alkoxides of alkali metals and/or alkaline earth metals and of alkali metal and/or alkaline earth metal salts, tin salts and of mixed metal oxides. Particularly suitable hydroxides of alkali metals and/or alkaline earth metals are lithium hydroxide and/or magnesium hydroxide. Within the group of oxides of alkali metals and/or alkaline earth metals, the oxides of magnesium are preferred. Preferred alkoxides of the alkali metals and/or alkaline earth metals are those which are derived from short-chain alcohols, for example those containing 1 to 8 carbon atoms, more particularly from methanol, ethanol and/or 2-ethyl hexanol. Magnesium and/or barium compounds are particularly preferred. Within the group of alkali metal and/or alkaline earth metal salts, the magnesium and barium salts, for example the carbonates, such as magnesium carbonate, or the acetates, for example magnesium acetate, are particularly important. Mixed metal oxides are oxide compounds which contain at least two different metals. One of the metals is preferably magnesium. The other metal may be aluminium, gallium, zirconium, indium, thallium, cobalt, scandium, lanthanum and/or manganese. Magnesium/aluminium mixed oxides are particularly preferred. The mixed oxides may be surface-modified with one or more of the co-catalysts already mentioned, more particularly with the hydroxides and/or alkoxides of the alkali metals and/or alkaline earth metals. Mixed metal oxides such as these and ways of modifying them are described, for example, in DE-A-44 46 064. Magnesium oxide is a particularly preferred co-catalyst. If the optionally modified hydrotalcites are sole catalysts, they are used in quantities of normally 0.1 to 5% by weight and preferably 0.5 to 1.5% by weight, based on starting compounds (compounds containing active hydrogen or carboxylic acid esters and alkylene oxide). If the optionally modified hydrotalcites are used together with the selected co-catalysts, they are used in quantities of normally 0.05 to 2.5% by weight and more particularly 0.1 to 0.5% by weight, based on the starting compounds. The co-catalysts may be used in quantities of 0.05 to 5% by weight, preferably in quantities of 0.1 to 0.5% by weight and more particularly in quantities of 0.1 to 0.3% by weight, based on the starting compounds. By virtue of the synergistic effect in catalyst activity between optionally modified hydrotalcite and the co-catalysts, it is even possible in accordance with the invention to obtain very good results with a total quantity of hydrotalcite and co-catalysts below 0.5% by weight, preferably with quantities of 0.1 to 0.4% by weight and more particularly with quantities of 0.2 to 0.3% by weight, based on the starting compounds. The ratio between optionally modified hydrotalcite as catalyst and co-catalysts may vary within wide limits and is preferably between 5:1 to 1:5, more preferably between 3:1 and 1:3 and most particularly between 2:1 and 1:2. The reaction of the compounds containing active hydrogen atoms or the carboxylic acid esters with the alkylene oxides may be carried out in known manner at temperatures of 120 to 200° C. and preferably 150 to 180° C. and under pressures of 1 to 5 bar. The quantity of alkylene oxide to be added on is not critical and may amount, for example, to between 1 and 100, preferably to between 2 and 50 and more particularly to between 2 and 20 moles of alkylene oxide per mole of H-active compound or carboxylic acid ester. Ethylene, propylene and/or butylene oxide may be used as the alkylene oxide, ethylene oxide being preferred. Now, it is crucial to the invention that, after the actual alkoxylation, the catalysts used, namely the optionally modified hydrotalcite on its own or in admixture with the selected co-catalysts, are completely decomposed in the reaction mixture obtained by the addition of acids. Both inorganic and organic acids may be added as the acids. Suitable inorganic acids are, in particular, the mineral acids, such as sulfuric acid, hydrochloric acid and/or phosphoric acid. Suitable organic acids are both the so-called fatty acids containing 6 to 22 carbon atoms and lower carboxylic acids containing 1 to 4 carbon atoms (only the carbon atoms of the hydrocarbon chain are counted, not the carbon atoms of the carboxyl groups) which may optionally be additionally modified with hydroxyl groups. Preferred organic acids are the lower carboxylic acids, such as lactic acid, acetic acid and/or citric acid. The acids are generally added as aqueous solutions, preferably as 10 to 90% by weight solutions. According to the invention, the acids are used in at least equimolar quantities, based on hydrotalcite and any co-catalysts present. The actual quantity of acid added is of course dependent on the strength of the acid. Molar ratios of acid to hydrotalcite and co-catalysts, if any, of generally 1:1 to 10:1 and more particularly 1:1 to 4:1 are recommended. In one advantageous embodiment of the invention, the acids are added at temperatures above the melting point of the alkoxylated nonionic surfactants, preferably at temperatures of 70 to 95° C. In practice, this is best done by keeping the reaction mixture obtained after the alkoxylation at those temperatures and adding the acid. The effect of adding the acid is that the hydrotalcite decomposes. The decomposition products of the hydrotalcite are soluble in water and do not lead to any clouding or sedimentation of the alkoxylated nonionic surfactants. Accordingly, there is no need in the process according to the invention for the elaborate filtration of the optionally modified hydrotalcite. If desired, the acid treatment according to the invention may of course be followed by working up of the water-soluble decomposition products of the hydrotalcite, for example by separation of the aqueous phasefrom the organic phase. The present invention also relates to the use of acids for decomposing optionally modified hydrotalcite and co-catalysts present as catalyst in reaction mixtures of alkoxylated nonionic surfactants. The alkoxylated nonionic surfactants obtained by the process according to the invention may be used without further filtration. No precipitation or sedimentation occurs, even after prolonged storage. Accordingly, they are suitable as nonionic surfactants for the production of laundry detergents, dishwashing detergents and cleaners and for the production of cleansing cosmetics, more particularly liquid products, such as liquid laundry detergents, hair shampoos and the like. EXAMPLES Example 1 288.6 g (=1.35 mole) of a methyl laurate were introduced into a pressure reactor together with 5.0 g (=0.5% by weight, based on starting compounds) of calcined hydrotalcite. The reactor was evacuated for 30 minutes at 100° C. and then purged with nitrogen. 711.4 g (=12 moles) of ethylene oxide were added in portions at max. 180° C. and max. 5 bar pressure. The reaction time was 150 minutes. After the alkoxylation, the reaction mixture was after-reacted for 1 hour at 120° C. and the reactor was evacuated for another 30 minutes at 120° C. 100 g of a 10% by weight citric acid were added to this reaction mixture with stirring at 90° C. The calcined hydrotalcite had dissolved after stirring for 10 minutes. The reaction mixture obtained was clear in the melt. Example 2 A mixture of 65.3 g (=0.41 mole) of a caprylic acid methyl ester and 261.3 g (=1.12 mole) of a methyl ester of palm kernel oil (fatty acid chain containing 12 to 18 carbon atoms) was introduced into a pressure reactor together with 1.0 g (=0.1% by weight, based on starting compounds) of calcined hydrotalcite and 2.0 g of magnesium oxide (=0.2% by weight, based on starting compounds). The reactor was evacuated for 30 minutes at 100° C. and then purged with nitrogen. 673.4 g (=10 moles) of ethylene oxide were added in portions at max. 180° C. and max. 5 bar pressure. The reaction time was 160 minutes. After the alkoxylation, the reaction mixture was after-reacted for 1 hour at 120° C. and the reactor was evacuated for another 30 minutes at 120° C. 68.4 g of a 20% by weight acetic acid were added to this reaction mixture with stirring at 90° C. The catalyst mixture of hydrotalcite and magnesium oxide dissolved completely in three minutes. Example 3 638.3 g (=3,3 moles) of a C 12/14 fatty alcohol mixture (ca. 70% by weight C 12 alcohol and ca. 30% by weight C 14 alcohol) were introduced into a pressure reactor together with 1.0 g (=0.1% by weight, based on starting compounds) of calcined hydrotalcite and 2.0 g of magnesium oxide (=0.2% by weight, based on starting compounds). The reactor was evacuated for 30 minutes at 100° C. and then purged with nitrogen. 361.7 g (=8.2 moles) of ethylene oxide were added in portions at max. 180° C. and max. 5 bar pressure. The reaction time was 70 minutes. After the alkoxylation, the reaction mixture was after-reacted for 1 hour at 120° C. and the reactor was evacuated for another 30 minutes at 120° C. An ethoxylated C 12/14 fatty alcohol with a hydroxyl value of 179 was obtained. 11.4 g of a 20% by weight acetic acid was added to the reaction mixture obtained with stirring at 90° C. The hydrotalcite and the magnesium oxide had dissolved completely after 15 minutes. | Summary: Processes for preparing alkoxylated nonionic surfactants are disclosed, wherein a compound having at least one active hydrogen atom, a carboxylic acid ester or mixtures thereof are reacted with an alkylene oxide in the presence of a hydrotalcite catalyst to form a reaction product mixture; and the reaction product mixture is combined with an acid in at least an equimolar amount based on the amount of the catalyst present. | 4,494 | 97 | big_patent | en |
Summarize: This application is a continuation in part of my now pending U.S. patent application Ser. No. 09/921,498 filed on Aug. 2, 2001. BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates generally to the field of wheelchairs and, more specifically, to an electrical braking system and quick release, detachable wheels for manual wheelchairs. 2. Description of the Related Prior Arts Numerous types of braking mechanisms for manual wheelchairs are known in the art. The most typical manual wheelchair brake is a manual “over center” locking device which is activated by a lever arm and, when forced into its locking position, presses a braking member against the surface of the wheelchair tire creating a frictional braking action. Several factors mitigate against the usefulness and reliability of these types of brakes. Loss of tire pressure reduces the frictional force exerted by the crossbar on the tire and hence reduces the braking effect. A significant air pressure loss leaves these brakes useless. During transfer in and out of the chair, this type of brake allows the tire to slide underneath the crossbar and the wheelchair to move. Similarly, the brakes are ineffective and will not adequately hold the wheelchair on an incline. Other types of manual brakes include caliper type brakes manually activated with a lever arm mounted to a cable and brake assembly causing brake pads to press against the rim of the wheelchair wheel. In these types of brakes, the frictional braking force exerted is directly related to the manual force which must be exerted on the lever arm by the brake operator to activate the brake. Wheelchair users who have arm or hand limitations may not be physically able to operate these brakes. These braking mechanisms only apply a braking force to one wheel. If an equal braking force is desired on both wheels, the user is required to use both arms and attempt to apply an equal force to both lever arms at the same time. This is difficult, if not impossible. Wheelchair frame and wheel design most often require the placement of the lever arms on the frame of the wheelchair near the user's knees. The placement of these lever arms interferes with the user's transfer in and out of the wheelchair. These lever arms require lifting the user's body in order to clear the lever during transfer. A patent to Ross and Gunther, U.S. Pat. No. 5,358,266 describes a plate attached to a braking member, which applies a braking frictional force to the wheelchair tire when electronically activated by a solenoid rod. The solenoid rod is activated by means of a switch attached to the seat of the wheelchair. When the wheelchair user is raised out of the seat, the switch is activated and operates the braking mechanism. Also disclosed in this patent is a manually activated lever arm to operate the same braking member when the wheelchair user is seated. The same deficiencies discussed above apply to this wheelchair while the wheelchair user is seated. A wheelchair user with arm or hand limitations may not be able to operate the hand lever and the lever arm braking mechanism to apply a braking force to one wheel. In addition, the position of the lever arm may interfere with transfer in and out of the wheelchair. Electric wheelchairs with various forms of braking means are common in the prior art. These braking means include gear reduction mechanisms, electromagnetic braking by means of a resistance applied to the electric motors, electronically activated frictional braking mechanisms where a solenoid is electrically energized to move brake shoes into frictional contact with a brake drum, and conventional manual brakes operated by a lever mechanism. These electric wheelchairs are heavy, cumbersome, difficult to transport, and do not promote physical activity by the user. Wheelchair users have reason to frequently remove the wheels from their wheelchairs. It is often done for storage purposes, for brake adjustment, for wheel repair, and for wheel exchange. For example, in order to store a wheelchair in a vehicle, it is often desirable to remove the wheels. Heretofore, the wheels on manual wheelchairs and other types of wheelchairs have been attached to the wheelchair frame by some type of hub with the wheels secured to the hub with nuts and bolts. In order to remove the wheels from the wheelchair, it has been necessary to unscrew and remove each of the nuts and bolts securing the wheel to the hub. This is a time consuming and cumbersome process. Once again, wheelchair users who have arm or hand limitations may not be physically able to remove the nuts and bolts. More recently, it has become common in the art to attach wheels to manual wheelchairs using quick release locking pins which hold the wheel to the axle. In this type of design, it is difficult to also have a braking means on the wheelchair wheel other than the manual “over center” locking device which presses a braking member against the surface of the tire as described herein. Heretofore, other brakes have been ineffective on wheelchairs with quick release locking pins because the braking means had to be released and moved or disassembled in order to remove the wheel and thereby defeating the purpose of the quick release locking pin. It is desirable to have a lightweight, manual wheelchair with an effective easily operatable electronic braking mechanism and, at the same time, quick release detachable wheels. SUMMARY OF THE INVENTION It is an object of this invention to provide an electronically activated braking system for a lightweight, manual wheelchair, which allows the wheelchair to maintain its lightweight and maneuverability characteristics. It is a further object of this invention to have an electronically activated braking system for manual wheelchairs which eliminates the need for users of the wheelchair to manually operate brakes by means of a lever mechanism. It is a further object of this invention to provide a braking system for manual wheelchairs, which provides equal braking force to both wheels of a wheelchair simultaneously. It is a further object of this invention to provide a braking means for a manual wheelchair, which can be activated without the use of a manually operated lever that interferes with transfer in and out of the wheelchair by the user. It is a further object of this invention to provide a braking means for manual wheelchairs, which eliminates movement of the wheelchairs on inclines and during transfer in and out of the wheelchair by the user. It is a further object of this invention to provide a braking means for manual wheelchairs, which allows for detaching the wheelchair wheels without disturbing the braking means. It is a further object of this invention to provide for quick release, easily detachable wheels. It is a further object of this invention to provide for detachable wheels, which eliminates the need for users of the wheelchair to unscrew numerous nut and bolt combinations in order to remove the wheel. It is a further object of this invention to provide for quick release, easily detachable wheels which allow the wheels to be removed without removing the disk and brake assembly. In order to achieve these objectives, this invention provides for an electronic braking system, which is comprised of a braking means, a cable pulley system for activating the braking means, a DC liner actuator with actuator rod connected to the cable pulley system, a motion limit switch, a rechargeable twelve-volt battery electronically connected to the DC actuator, and a double throw control switch electronically connected to the battery for activating the battery power. It is anticipated that the preferred braking means is a caliper-type brake positioned to clamp onto a metal disk mounted axially to a hub which rotates on the axle of each wheelchair wheel. The hub on which the disk is mounted interlocks with the hub on which the wheelchair wheel is mounted. The interlocking hubs are locked together with a locking pin, which extends axially through the center of the mated hubs such that the hubs are locked and rotate together when the wheelchair wheel is turned. The locking pin is equipped with retractable nipples which, when extended, hold the locking pin securely in place. The retractable nipples are spring biased in the extended position and are activated by a push button at one end of the locking pin which releases the spring and allows the nipples to retract. When the nipples are in the retracted position, the locking pin can be removed simply by sliding it out of the axle. This allows the wheelchair wheel to be removed since there is no longer anything holding the mated hubs together. The braking means for each wheel are connected to opposite ends of a cable wire. The cable wire passes around a pulley such that displacement of the pulley provides equal force and displacement to said opposite ends of the cable wire. The ends of the cable wire are directed through small openings in a mounting bracket. The openings are spaced a distance equal to the diameter of the pulley so the cable wire remains parallel as it extends from the pulley through said openings. A circular pulley cap is placed concentrically over the pulley. The vertical side of the pulley cap has two openings to allow for the passage of the wire cable into the pulley cap through the first opening, around the pulley and out the second opening. The pulley cap, pulley, and cable wire assembly is then connected to the outer end of the actuator rod by a coupling bracket. The DC linear actuator is mounted on the wheelchair in a manner to allow the actuator rod to extend and displace the pulley and cable wire in line with the actuator rod's axis. The DC linear actuator is electronically powered by a twelve-volt rechargeable battery mounted to the wheelchair. The battery power is activated by a double throw control switch mounted to the wheelchair in a position where it is easily accessed by both the wheelchair user and a person assisting the wheelchair user. The double throw toggle switch can be thrown in two different directions. When the double throw toggle switch is thrown in the first direction, it will cause the actuator rod to retract, pulling the pulley and cable wires and activating the braking force. When the toggle switch is thrown in the second direction, it will cause the actuator rod to extend, pushing the pulley and cable wire and deactivating the braking force. In order to limit the tension in the cable wire, a motion limit switch can be added to the electrical brake system. The motion limit switch is wired into the circuit between the double throw toggle switch and said DC linear actuator. The motion limit switch is activated by displacement of the actuator rod in the direction which pulls the cable wire and activates the braking means. Once a selected braking force is attained, the motion limit switch opens the circuit and stops the displacement of the actuator rod. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a elevational side view of a manual wheelchair depicting a caliper braking mechanism mounted to the wheelchair frame and positioned to clamp onto a metal disk mounted axially to the hub of the wheelchair wheel. FIG. 2A is an enlarged exploded perspective view depicting the locking pin, wheelchair wheel, hub, disk, and axle assembly which has a spring biased push button type locking pin and first interlocking hub design. FIG. 2B is an enlarged exploded perspective view depicting the locking pin, wheelchair wheel, hub, disk, and axle assembly wherein the locking pin is equipped with a lever which activates an expandable tip. FIG. 2C is an enlarged exploded perspective view depicting FIG. 2A from the opposite angle. FIG. 2D is an enlarged exploded perspective view depicting the locking pin, wheelchair wheel, hub, disk, and axle assembly. This figure depicts a second interlocking hub design. FIG. 2E is an enlarged exploded perspective view depicting FIG. 2D from the opposite angle. FIG. 3 is a bottom view of the wheelchair seat depicting the toggle switch, the battery recharging outlet, the electrical wiring, the twelve-volt rechargeable battery, the DC linear actuator, the cable wire and pulley assembly, and the motion limit switch. FIG. 4 is an enlarged perspective view depicting the caliper braking mechanism. FIG. 5 is an exploded perspective view depicting the cable wire and pulley assembly and actuator rod mount. FIG. 6 is a bottom view of the cable wire, pulley, and actuator rod assembly brackets and the motion limit switch. FIG. 7 is a elevational side view of the coupling bracket. FIG. 8 is an electrical circuit diagram illustrating the electrical control circuit of this invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT Referring to FIG. 1, a lightweight manual wheelchair 10 is equipped with a solid seat base 11, seat cushion 12, and seat back 13 mounted between first and second wheelchair wheels 24 generally to a frame 14. The frame 14 has a vertical component 15, a side horizontal component 16, a frontal curved component 17 and a lower curved component 20. A footrest 19 is mounted at the frontal extremity of the lower curved component 20 of the frame 14. First and second caster wheels 21 are pivotally mounted toward the frontal extremity of the lower curved component 20 of the frame 14. The manual wheelchair 10 is symmetrical about a centre line and the opposed side is identical to the side visible in FIG. 1. Thus, when the first and second of numbered items are referred to without the second item being shown, it can be appreciated that the second numbered item is identical to the first but on the opposite side of the wheelchair. First and second caliper brakes 18 are mounted to extension plates (not shown) which are in turn mounted to the frame 14. The caliper brakes 18 are positioned to clamp onto first and second disks 22 (see FIGS. 1 and 4 ). In the preferred embodiment of this invention, the first and second caliper brakes 18 are manufactured by Hayes/HMX, model number BR3920. However, numerous other cable actuated caliper brakes are available on the market and can be used in this invention. The first and second wheelchair wheels 24 can be detached without removal of the first and second disks 22 or the first and second caliper brakes 18. Referring to FIGS. 2A through 2E, the first and second disks 22 are concentrically mounted to the inner face 83 first and second disk hubs 23 by means of a plurality of screws 29 passing through radially spaced interiorly threaded, aligned holes 51 in the first and second disk hubs 23 and the first and second disks 22. In the preferred embodiment, as shown in FIGS. 2A, 2 B, and 2 C, the screws 29 are Allen screws where the heads 33 of the screws 29 extend from the outer vertical faces 27 of the first and second disk hubs 23 and are secured on the opposite end by nuts 38. In a second preferred embodiment, as shown in FIGS. 2D and 2E, the screws 29 are of a length insufficient to extend beyond the outer vertical faces 27 of the first and second disk hubs 23. The first and second disk hub 23 and disk 22 assemblies are concentrically mounted to outer ends of first and second detachable axle pieces 80 and rotate thereon. The first and second detachable axle pieces 80 are tubular with a smooth surface portion 82 at their outer end and a exteriorly threaded portion 84 at their inner end. The smooth surface portion 82 and the exteriorly threaded portion 84 are divided by a flange 86. The first and second detachable axle pieces 80 are mounted to the frame 14 of the wheelchair 10 (see FIG. 1) by screwing the exteriorly threaded portion 84 into a tubular axle 25. As shown in FIG. 3, the tubular axle 25 is clamped to the first and second lower curved components 20 of the frame 14 (See FIG. 1) at its rear extremity by first and second frame clamps 72. Referring again to FIGS. 2A through 2E, the outer ends of the tubular axle 25 have mounting heads 88. Each mounting head 88 has a threaded bore 90 with a diameter sufficient to accept and secure the exteriorly threaded portion 84 of the first and second detachable axle pieces 80 therein. The first and second detachable axle pieces 80 are mounted to the tubular axle 25 by screwing the exteriorly threaded portion 84 into the threaded bore 90. The first and second disk hub 23 and disk 22 assemblies are secured to the first and second detachable axle pieces 80 by means of a clip ring 39. The clip ring 39 is spring biased to close around and fit in to a circumferential groove 78 cut into the smooth surface portion 82 of the first and second detachable axle pieces 80 at their extreme outer end. In order to allow the first and second disk hub 23 and disk 22 assemblies to rotate on the first and second detachable axle pieces 80, the smooth surface portion 82 of the first and second detachable axle pieces 80 extend axially through a tubular opening 92 at the center of the first and second disk hubs 23 and the outer face of flange 86 abuts a concentric circular shoulder 87 (see FIGS. 2C and 2D) on the inner face 83 of the first and second disk hubs 23 with a spacer ring 94 between. The spacer ring 94 prevents frictional contact between the outer face of flange 86 and the circular shoulder 87 on the inner face of the first and second disk hubs 23. In the preferred embodiment, the spacer ring 94 is a Delrin washer. However it is anticipated that other smooth, durable material can be substituted. Referring to FIGS. 2A, 2 B, and 2 E, the outer vertical face 27 of the first and second disk hub have a concentric circular recessed portion 93 surrounding the tubular opening 92. The horizontal length of the smooth surface portion 82 of the detachable axle piece 80 is sufficient to allow the smooth surface portion 82 to extend through the tubular opening 92 of the first and second disk hubs 23 and expose the circumferential groove 78 on the opposite side of the first and second disk hubs 23 with minimal clearance at the concentric circular recessed portion 93. This allows the clip ring 39 to close around circumferential groove 78 within the concentric circular recessed portion 93. As shown in FIGS. 2A through 2C, the first and second wheelchair wheels 24 are concentrically mounted on the first and second wheel hubs 37. The inner surface 57 of the first and second wheelchair wheels 24 (See FIG. 2C) is mounted flush against the outer vertical surface 70 (See FIG. 2E) of the flanged inner portion 31 of the first and second wheel hubs 37 and are secured to the first and second wheel hubs 37 by first and second nuts 45, which screw onto exteriorly threaded outer ends 75 of the first and second wheel hubs 37. The first and second wheel hubs 37 have a tubular opening 43 through their center. As shown in FIGS. 2A and 2B, an outer circular bearing assembly 61 is pressed fit into the tubular opening 43 towards the outer end of the first and second wheel hubs 37. As shown in FIGS. 2B, 2 C, and 2 D, an inner circular bearing assembly 79 is pressed fit into the tubular opening 43 at the inner end of the first and second wheel hubs 37. The outer bearing assembly 61 and inner bearing assembly 79 have inner rings 63 which turn within the bearing assemblies. The inner diameter of the inner rings 63 is equal to the inner diameter of first and second detachable axle pieces 80. In the preferred embodiment, the outer circular bearing assembly 61 and inner circular bearing assembly 79 are manufactured by NICE, Model No. 1616 DC TN or KYK, Model No. R-8-DDHA1(IB). However, it is anticipated that other similar bearings could be used. Referring again to FIGS. 2A through 2E, when the first and second wheelchair wheels 24 are mounted to the wheel hub 37 and in turn mounted to the wheelchair 10 (See FIG. 1 ), the outer vertical faces 27 of the first and second disk hubs 23 interlock with inner faces 77 of the flanged inner portion 31 of the first and second wheel hubs 37. In the preferred embodiment, as shown in FIGS. 2A, 2 B, and 2 C, the inner faces 77 of the flanged inner portion 31 of the first and second wheel hubs 37 are flat with a plurality of radially spaced holes 96 shown in FIG. 2 C. The heads 33 of the plurality of screws 29 fit snugly into the corresponding radially spaced circular holes 96 in the flanged inner portion 31 of the first and second wheel hubs 37. In an alternate embodiment, as shown in FIGS. 2D and 2E, the inner face 77 of the flanged inner portion 31 of the first and second wheel hubs 37 have a raised surface 98 extending from the inner face 77. The raised surface 98 is centered on the inner face 77 with parallel sides 100 extending to the circumference of the inner face 77. The parallel sides 100 extend perpendicularly from the inner face. In this alternate embodiment, the outer vertical faces 27 of the first and second disk hubs 23 have a channel 102. The placement and dimensions of the channel 102 are to allow the raised surface 98 to fit snugly into the channel 102 with minimal clearance at all contiguous surfaces when the first and second wheel hubs 37 are interlocked with the first and second disk hubs 23. In the preferred embodiment, as shown in FIGS. 2A, 2 B, and 2 C, the interlocking of heads 33 within the radially spaced circular holes 96 cause the first and second wheelchair wheels 24 and the first and second disks 22 to rotate together. In another alternate embodiment, as shown in FIGS. 2D and 2E, the interlocking of the raised surface 98 on the inner face 77 of the first and second wheel hubs 37 with the channel 102 in the outer vertical faces 27 of the first and second disk hubs 23 cause the first and second wheelchair wheels 24 (See FIG. 1) and the fist and second disks 22 to rotate together. Still referring to FIGS. 2A through 2E, in order to hold the first and second disk hubs and the first and second wheel hubs together when interlocked, first or second locking pins 35 a and 35 b (see FIGS. 2A and 2B) extend axially through the center of the first and second wheel hubs 37, the first and second disk hubs 23, and into the first and second detachable axle pieces 80. The first or second locking pins 35 a and 35 b have a diameter which allows the first or second locking pins 35 a and 35 b to slide through the inner rings 63 of the outer circular bearing assembly 61 (See FIGS. 2A and 2B) and the inner circular bearing assembly 79 (See FIGS. 2C and 2D) and into the first and second detachable axle pieces 80 with minimal clearance. The first and second wheelchair wheels 24 can be detached from the wheelchair 10 (See FIG. 1) without removing the first and second disks 22 or disturbing the first and second caliper brakes 18 by removing the first and second locking pins 35 a or 35 b and separating the first and second wheel hubs 37 from the first and second disk hubs 23. In the preferred embodiment of the invention (see FIGS. 2A, 2 C, 2 D, and 2 E), the first and second locking pins 35 a have a push button 47, a rod 49, an adjusting nut 53, and a set of retractable nipples 55. The push button 47 is spring biased in the released position, causing the retractable nipples 55 to extend from the rod 49. When the push button 47 is depressed, the retractable nipples 55 retract into the rod 49. The first and second locking pins 35 a can be inserted through the inner ring 63 of the outer circular bearing assembly 61 and into the tubular openings 43 of the first and second wheel hubs 37 by depressing the push button 47 and thereby causing the retractable nipples 55 to retract. When the first and second locking pins 35 a are further inserted through the first and second disk hubs 23 and into the first and second detachable axle pieces 80 and the push button 47 is released, the retractable nipples 55 extend into grooves (not shown) circumferentially cut into the tubular interior surface (not shown) of the first and second detachable axle piece 80. The grooves (not shown) are of sufficient depth and width to allow the retractable nipples 55 to extend into the grooves (not shown) with minimal clearance. The grooves (not shown) are positioned in the first and second detachable axle pieces 80 to allow the retractable nipples 55 to extend into the first and second grooves (not shown) when the first and second locking pins 35 a are fully inserted into the first and second wheel hubs 37 such that the adjustable nut 53 contacts the outer surface of the outer circular bearing assembly 61. In the preferred embodiment, the first and second locking pins 35 a are QRP Quick Release Push Button (large/small) Axle, Model No. 21QRP11CDASN. In an alternate embodiment of the invention, the length of the exteriorly threaded portion 84 of the first and second detachable axle pieces 80 is sufficient to allow the position of the retractable nipples 55 on the first and second locking pins 35 a to extend beyond the inner lip 85 of the first and second detachable axle pieces 80 when the first and second locking pins 35 a are fully inserted into the first and second wheel hubs 37 such that the adjustable nut 53 contacts the outer surface of the outer circular bearing assembly 61. Thus, when the first and second locking pins 35 a are fully inserted and the push button 47 is released, the retractable nipples 55 extend adjacent to the inner lip 85 of the first and second detachable axle pieces 80 with minimal clearance and thereby holding the first and second locking pins 35 a in place. In this embodiment, the first and second locking pins 35 a are, once again, QRP, Quick Release Push Button (large/small), Axle Model No. 21QRP11CDASN. In yet another embodiment of the invention (see FIG. 2 B), the first and second locking pins 35 b have a release lever 65 at one end of a rod 67, a spacer joint 69 between the release lever 65 and the rod 67, an expandable tip 71 attached to the other end of the rod 67, and a wedging cap 73 attached to the expandable tip 71 opposite the rod 67. When the release lever 65 is rotated to the released position so that it extends parallel with the rod 67, the diameter of the expandable tip 71 is not expanded and is equal to the diameter of the rod 67. When the release lever 65 is rotated perpendicular to the rod 67, the wedging cap 73 is pulled toward the release lever 65 causing the expandable tip 71 to expand to a diameter greater than the diameter of the rod 67. When the release lever 65 is in the released position, the first and second locking pins 35 b can be inserted through the inner ring 63 of the outer circular bearing assembly 61 and into the tubular opening 43 of the first and second wheel hubs 37. When the first and second locking pins 35 b are inserted through the first and second wheel hubs 37, and into the first and second detachable axle pieces 80 and the release lever 65 is then rotated perpendicular to the rod 67, the expandable tip 71 expands into and makes frictional contact with the interior surface (not shown) of the first and second detachable axle pieces 80. The frictional force created is great enough to hold the first and second locking pins 35 b in place. The diameter of the spacer joint 69 is greater than the inner diameter of the inner ring 63 of the outer circular bearing assembly 61, such that when the first and second locking pins 35 b are fully inserted, the spacer joint 69 contacts the outer face of the outer circular bearing assembly 61. In this preferred embodiment, the locking pin 35 b is the Ultra Axle, 0.50″ O.D. manufactured by Rousson Chamoux. The first and second caliper brakes 18 are activated by pulling a cable wire 26 (See FIGS. 4 and 5) attached to the caliper brakes 18 at first and second ends of the cable wire 26. The first and second ends of the cable wire 26 are directed to the first and second caliper brakes 18 through a cable wire housing 28 which is attached to a nozzle 30 on the first and second caliper brakes 18. The first and second ends of the cable wire 26 are attached to the first and second caliper brakes 18, respectively, in typical fashion. The cable wire 26 passes through the nozzle 30 of the first and second caliper brakes 18 and into the cable wire housing 28. The cable wire housing 28 directs the cable wire 26 to a mounting bracket 32 (See FIG. 5 ). The mounting bracket 32 has a vertical portion, and an upper horizontal portion. The mounting bracket 32 is mounted to the bottom of the solid seat base 11 by two screws (not shown) passing through interiorly threaded aligned holes in the solid seat base 11 and upper horizontal portion of the mounting bracket 32. The cable wire housing 28 is connected to the mounting bracket 32 by means of first and second hollow connectors 34. The first ends of the first and second hollow connectors 34 fit snugly within first and second circular openings (not shown) in the mounting bracket 32 and the second ends of the first and second hollow connectors 34 fit snugly around the cable wire housing 28. The centers of said first and second circular openings (not shown) are equidistant from the upper horizontal portion of the mounting bracket 32 and are horizontally spaced a distance equal to the diameter of the pulley 36. The diameter of the first and second circular openings (not shown) is sufficient to allow the first and second hollow connectors 34 to fit snugly and the cable wire 26 to pass through first and second circular openings (not shown) within the first and second hollow connectors 34. The cable wire 26 passes through the circular openings in the mounting bracket 32 within the first and second hollow connectors 34 and then passes around the pulley 36. The pulley 36 and cable wire 26 assembly is covered with a circular pulley cap 40. The inner diameter of the circular pulley cap 40 is of sufficient dimension to cover the pulley 36 and wire cable 26 assembly with minimal clearance. The vertical side of the pulley cap 40 has first and second openings 41 spaced to allow the cable wire 26 to pass into the pulley cap 40 and around the pulley 36. In the preferred embodiment of this invention, the segments of the cable wire 26 on opposite sides of the pulley 36 between the pulley 36 and mounting bracket 32 are parallel. Both segments of the cable wire 26 are perpendicular to the vertical side of the mounting bracket 32. The pulley cap 40, pulley 36, and wire cable 26 are connected to an actuator rod 42 of a DC linear actuator 50 (See FIG. 3) by means of a coupling bracket 44. The pulley cap 40, pulley 36, and wire cable 26 are connected to the coupling bracket 44 by a bolt and nut combination 46 passing through holes vertically aligned with the axis of the pulley cap 40 and pulley 36. The actuator rod 42 is connected to the coupling bracket 44 by a bolt and nut combination 48 passing through holes horizontally aligned through the coupling bracket 44 and through the center of the outer end of the actuator rod 42. The DC linear actuator 50, as shown in FIG. 3, is mounted to the solid seat base 11 by means of a mounting flange 56 and an actuator mounting piece 52. The actuator mounting piece 52 is mounted to the solid seat base 11 by two nut and bolt combinations. The mounting flange 56 is mounted to the actuator mounting piece 52 by a nut and bolt combination passing through horizontally aligned holes in the mounting flange 56 and first and second vertical portions 54 of the actuator mounting piece 52. The DC linear actuator is positioned so that displacement of the actuator rod 42 is in a direction perpendicular to the vertical portion of the mounting bracket 32 and centered between the first and second circular openings (not shown) in the vertical portion of the mounting bracket 32. In the preferred embodiment, the DC linear actuator 50 is manufactured by Warner Electric, model number DE12Q17W41-02FHM3HN. The DC linear actuator 50 is powered by a twelve-volt rechargeable battery 58 mounted to the bottom of the solid seat base 11. In the preferred embodiment of this invention, the twelve volt rechargeable battery 58 is mounted to the solid seat base 11 by first and second Velcro straps 59. Each of the first and second Velcro straps 59 pass through two slits (not shown) in the solid seat base 11 such that each of the first and second Velcro straps 59 pass through the first slit (not shown) to the top of the solid seat base 11 and back through the second slit (not shown) and around the twelve volt rechargeable battery 58. In the preferred embodiment of this invention, the twelve volt rechargeable battery 58 is a sealed, non-spillable, lead battery manufactured by CSB Battery Company, Ltd. A recharger outlet 68 is mounted to the frame 14 and is wired across the positive and negative leads of the twelve volt rechargeable battery 58. In the preferred embodiment of this invention, the recharger outlet 68 is mounted to the rear of the solid seat base 11. However, the recharger outlet 68 can be mounted generally to any part of the frame 14 where it is convenient and accessible. As shown in FIGS. 3 and 8, the battery power is controlled by a double throw toggle switch 60 which is mounted to the frame 14. In the preferred embodiment of this invention, the double throw toggle switch 60 is mounted to vertical component 15 of the frame 14. (See FIG. 1.) However, the double throw toggle switch 60 can be mounted generally to any part of the frame 14 where it is convenient and accessible to the wheelchair user. The double throw toggle switch 60 is wired into the electrical circuit, as shown in FIG. 7, across the positive and negative leads of the twelve volt rechargeable battery 58. The double throw toggle switch 60 can be thrown in a first direction 74 or a second direction 76. If the double throw toggle switch 60 is thrown in the first direction 74, it closes the circuit and powers the motion of DC linear actuator 50 and causes the actuator rod 42 to retract. The retraction of the actuator rod 42 pulls the pulley 36 and cable wire 26 assembly causing the displacement of the cable wire 26 within the cable wire housing 28 in a direction away from the first and second caliper brakes 18 (See FIGS. 4, 5, and 6 in combination). The displacement of the cable wire 26 away from the first and second caliper brakes 18 causes equal tension in the cable wire 26 on opposite sides of the pulley 36 and activates the first and second caliper brakes 18 with equal braking force. If the double throw toggle switch 60 is thrown in the second direction 76, it closes the circuit and the polarity and direction of current flow through the DC linear actuator 50 is reversed. This powers the motor of the DC linear actuator 50 in the reverse direction and causes the actuator rod 42 to extend. The extension of the actuator rod 42 displaces the pulley 36 and causes the cable wire 26 to move within the cable wire housing 28 toward the first and second caliper brakes 18. This in turn releases the tension in the cable wire 26 created by retracting the activator rod and deactivates the first and second caliper brakes 18. The first and second caliper brakes 18 are spring biased (not shown) toward the deactivated position which retains tension in the cable wire 26 while the actuator rod 42 is extending and prevents bunching of the cable wire 26. In order to control the tension in the cable wire 26 when the actuator rod 42 is retracting, a motion limit switch 62 is placed in the electrical circuit, as shown in FIG. 7, between the positive lead of double throw toggle switch 60. When the double throw toggle switch 60 is thrown in the first direction 74, the motion limit switch 62 limits movement of the DC linear actuator 50. The motion limit switch 62 is equipped with a motion arm 64 as shown in FIGS. 3, 6, 7, and 8. The motion arm 64 is spring biased to contact and press against an actuating pin 66 as shown in FIGS. 3, 6, 7, and 8. The actuating pin 66 extends from, and is a part of, the coupling bracket 44 as more clearly illustrated in FIG. 6. The motion limit switch 62 is normally closed. Retraction of the actuator rod 42 causes displacement of the coupling bracket 44 and actuating pin 66, which in turn displaces the motion arm 64. Sufficient displacement of the motion arm 64 throws the motion limit switch 62 opening the circuit and preventing further retraction of the actuator rod 42. The displacement of the motion arm 64 required to throw the motion limit switch 62 is adjustable to allow for control and selection of the tension in the cable wire 26 and the resulting braking force. In the normal operation of the wheelchair 10, it is desirable to have brakes activated during the transfer in and out of the wheelchair 10. If the wheelchair user intends to transfer out of the wheelchair, he will throw the toggle switch 60 in the first direction 74 which causes the actuator rod 42 to retract and activates the first and second caliper brakes 18. The wheelchair user should hold the toggle switch 60 in the first direction 74, thereby increasing the braking force applied by the first and second caliper brakes 18 until the motion limit switch 62 is thrown and opens the circuit which stops the retraction of the actuator rod 42. The user should then release the toggle switch 60 which is spring biased to the center, OFF position. The motor of the DC linear actuator 50 locks the actuator rod 42 in position when there is no power to the DC linear actuator 50. Thus, the first and second caliper brakes 18 will remain activated and hold the wheelchair 10 in position while the wheelchair user transfers out of the chair. The first and second caliper brakes 18 will remain activated until the toggle switch 60 is thrown and held in the second direction 76 and thereby allowing the actuator rod 42 to extend a sufficient amount to deactivate the first and second caliper brakes 18 and allow the first and second wheelchair wheels 24 to rotate freely. The toggle switch 60 is then released allowing it to spring back to the center OFF position which opens the circuit and stops the flow of power to the DC linear actuator 50. Although the invention has been described with reference to specific embodiments, this description is not meant to be construed in a limited sense. Various modifications of the disclosed embodiments, as well as alternative embodiments of the inventions will become apparent to persons skilled in the art upon the reference to the description of the invention. It is, therefore, contemplated that the appended claims will cover such modifications that fall within the scope of the invention. | Summary: A quick release detachable wheel hub assembly is shown for a lightweight manual wheelchair. The wheelchair wheels mount on exterior hubs and rotate therewith. The inner face out of each of the exterior hubs mates with an opposing outer face of interior hubs. One of the opposing faces on the hubs has a projection or a plurality of projections which fit snugly into corresponding openings on the opposing face of the other hub when the opposing faces of the hubs are mated. The interior hubs are mounted and rotate on detachable axles which screw into the wheelchair frame. A quick release, removable locking pin is inserted through the center of the hubs and into detachable axle and locked in place and thereby causing the hubs to be locked and rotate together. The wheels are quickly detached by simply removing the locking pins and pulling apart the hubs. | 9,956 | 200 | big_patent | en |
Summarize: Il prossimo 5 gennaio, potremo vedere in sala “Assolo”, scritto (con Daniele Costantini), diretto ed interpretato dalla straordinaria Laura Morante. Si tratta della seconda prova registica della pluripremiata attrice toscana, dopo l’esordio con “Ciliegine”, commedia del 2012. In “Assolo”, la Morante è nei panni della protagonista, Flavia, donna estremamente insicura, comicamente imperfetta, con due matrimoni falliti alle spalle, due figli e facilmente “manipolabile” da tutti quelli che le stanno intorno. La sua vita si snoda tra situazioni surreali e comiche e blandi drammi esistenziali. Obiettivi principali da raggiungere? Prima di tutto l’autostima, poi la rinascita. A curare le musiche del film c’è Nicola Piovani e, stando alle immagini del trailer, la pellicola potrebbe rivelarsi una gradevole sorpresa al botteghino d’inizio anno. Flavia è una donna fragile e insicura. Ha due matrimoni alle spalle, due figli, un cane in prestito ed è sempre alla disperata ricerca del consenso e dell'affetto delle persone che la circondano. Incapace di separarsi emotivamente dai suoi ex mariti Gerardo e Willi, Flavia è in rapporti amichevoli anche con le loro nuove compagne, Giusi e Ilaria. In questa famiglia allargata Flavia è, però, sempre sola, incapace di raggiungere qualsiasi obiettivo, che sia la patente di guida o un corso di tango. Tra incidenti di percorso e sorprendenti scoperte, Flavia imparerà che nessuna donna è perfetta e che l’autostima e la libertà tanto inseguite erano proprio li, a portata di mano. | Summary: L'attrice toscana si cimenta nuovamente alla regia con una commedia in cui veste anche i panni della protagonista, Flavia, una donna insicura, comicamente imperfetta, con due matrimoni falliti alle spalle, due figli e facilmente "manipolabile" da tutti quelli che le stanno intorno. La sua vita si snoda tra situazioni surreali e blandi drammi esistenziali, in cerca dell'agognata autostima. | 369 | 97 | fanpage | it |
Summarize: Media caption The Holighost was one of Henry V's warships - which fought in battles against the French in the 15th century The wreck of a 600-year-old warship which helped Henry V wage war on France is believed to have been found buried in a river. The Holigost - or Holy Ghost - was one of four "great ships" commissioned by the king in his war against France. It was spotted in an aerial photograph by historian Dr Ian Friel, in an area of Hampshire's River Hamble described as a medieval breaker's yard. Historic England said it was a "tangible link" to Henry V. The Holigost fought in sea battles during the Hundred Years War which broke the French naval power. Dr Friel identified the wreck when he was revisiting documentary evidence for a book on Henry's navy. Image copyright PA Image caption The ship is thought to be buried in mud on the River Hamble Future scientific research on the ship, which could include sonar and aerial imaging using drones, could reveal much about 15th Century shipbuilding and improve understanding of life aboard ship, naval warfare of the time, dock building and docking practices. Historic England said it was taking steps to protect and investigate the shipwreck in part of the river next to where Henry's flagship, the Grace Dieu, was identified in the 1930s. Image copyright PA Holigost A major part of Henry V's war machine as he sought to conquer France Had a crew of 200 sailors and carried large numbers of soldiers to war The second of four "great" ships built for the king's royal fleet Underwater repair work on the ship carried out by a "dyver" called Davy Owen in 1423 may be the first-recorded example of a diver used in ship repair in England Duncan Wilson, Historic England's chief executive, said the investigation in the 600th anniversary year of the Battle of Agincourt, was "immensely exciting." "It holds the possibility of fascinating revelations in the months and years to come," he added. Dr Friel said: "In my opinion, further research leading to the rediscovery of the Holigost would be even more important than the identification of the Grace Dieu in the 1930s. "The Holigost fought in two of the most significant naval battles of the Hundred Years War, battles that opened the way for the English conquest of northern France." Advertisement Continue reading the main story LONDON — The heritage group Historic England announced Monday that it had discovered a timber ship it believes to be the 600-year-old wreck of an important vessel in King Henry V’s fleet. It says the ship, found in the mud of a river in southern England near Southampton, is likely to be the Holigost, or “Holy Ghost,” which played a major role in two sea battles that enabled Henry to conquer territory in France in the early 15th century, during the Hundred Years’ War. The announcement came just days before the 600th anniversary of the Battle of Agincourt. In that battle, on Oct. 25, 1415, Henry led his troops to a famous victory in northern France, described by Shakespeare in “Henry V.” If recovered safely and verified, the ship will be preserved and go on display in Portsmouth. Deep in the oozing mud of the river Hamble in Hampshire, visible only at the lowest tides as a U-shaped ripple on the surface, possibly lies a ship that was one of the glories of Henry V’s navy. Ian Friel, a historian who has devoted decades of research to Henry’s navy, believes it is the Holigost, built for the king in 1415 by recycling the hull of a captured Spanish warship, the Santa Clara. Not an inch now shows above the surface, but Friel – whose book on Henry’s navy is published on Monday – has convinced Historic England to commission work including sonar surveys of the seabed, drone photography of the site and possibly wood sample dating. The site is being considered for a protection order to defend it from treasure hunters, although it would have been stripped of any valuables when it was laid up. Most of Henry’s navy comprised hired-in ships or privately owned merchant ships pressed into service, but the Holigost was one of the four ‘great ships’ commissioned by the king. It was completed in November 1415, just too late to join the flotilla that ferried Henry, his soldiers and horses to the king’s victory at Agincourt in northern France, but suffered extensive damage in the thick of the fighting at the naval battle of Harfleur the following year, and was repaired in time to fight again in 1417. The ship was repaired again in 1423 by a diver called Davy Owen, the first recorded instance of a diver used to make underwater repairs. Henry himself may have sailed on it, as Friel found records of a royal cabin being added. The ship, built to terrorise and conquer the French, was painted with a French motto, Une sanz plus. “‘One and no more’ – in other words, the king alone is master,” said Friel. “Henry was making it perfectly clear – there’s God and there’s Henry, and that’s your lot. “It may have been the humbler ships that actually did most of the work, but these great ships were floating symbols of power and prestige, richly ornamented with elaborate carvings, flying huge flags, towering over the much smaller merchant ships. They would have been, and were intended to be, an absolutely awe-inspiring sight.” The ship was laid up in a specially built dock in the Hamble in the years after Henry’s death in 1422, and in its last years afloat was crewed by one man who had the unenviable responsibility of pumping and bailing day after day to keep the water out. Stripped of its mast, contents, and timbers from the superstructure including the cabins, it probably finally sank when the dock collapsed due to lack of maintenance, and has lain there ever since. Friel first spotted the site more than 30 years ago in a grainy aerial photograph that showed the wreck of a modern cabin cruiser, the known wreck of another of Henry’s ships and a peculiar shape in the mud, which the more he peered at it, the more it looked like the outline of half the hull of another ship. Facebook Twitter Pinterest Drawing of the Holigost. Photograph: Historic England/PA He and several archaeologists went out in a rubber dinghy and probed the mud with poles, enough to convince them there was something down there, but there was never funding for a full investigation. He stressed that publicising it now is not a publicity stunt for his book, but to ensure that the site gains protection. If he is right the Holigost lies close to the wreck of the most spectacular of Henry’s ships, the enormous Grace Dieu, the largest ship built in England up to that date, which proved a floating white elephant. Although that wreck was spotted in the 19th century, it was so enormous, with a keel 40 metres long – twice the size of Henry VIII’s Mary Rose built a century later, that it was thought modern, and only identified in the 1930s. It took so long to build, using more than 3,700 trees, that the sea war was over by the time it was finished. It apparently only sailed once, when the crew mutinied – possibly, says Friel, because it was so hard to sail – and forced it into the Isle of Wight. It was laid up within a few years without ever firing an arrow in anger – and was then hit by lightning, and burned to the waterline. Friel said: “The Holigost was never as spectacular as the Grace Dieu, but in some ways it is more important – an identifiable medieval ship, that fought in known engagements, would be an incredibly rare thing to find anywhere in the world.” | Summary: A British historian may have discovered the wreck of one of the four "great ships" built by King Henry V for his war on France six centuries ago, the BBC reports. "These great ships were floating symbols of power and prestige," Dr. Ian Friel tells the Guardian. "They would have been, and were intended to be, an absolutely awe-inspiring sight." Friel first noticed what could be the Holigost in an aerial photograph 30 years ago, but he lacked the funding to explore the wreckage, which is buried so deep in river mud that even at low tide all that can be seen is a U-shaped ripple. But heritage group Historic England announced the find Monday in the hopes of preserving the Holigost-if it is the Holigost-for display, the New York Times reports. According to the Times, the Holigost-or Holy Ghost-fought in two major battles that helped Henry conquer parts of France during the Hundred Years' War. And Henry himself may have sailed in it. At the end of its life, the Holigost sat unused with a single crew member charged with bailing out water every day to keep it from sinking, the Guardian reports. The great ship likely sank when its dock collapsed. Historic England hopes to use drone photography, sonar, and wood dating to verify the wreckage found in the River Hamble as the Holigost. It says further research on the ship could provide valuable information on 15th-century shipbuilding, ship life, naval strategies, and more, according to the BBC. | 1,829 | 348 | multi_news | en |
Write a title and summarize: Glutamate receptors are divided in two unrelated families: ionotropic (iGluR), driving synaptic transmission, and metabotropic (mGluR), which modulate synaptic strength. The present classification of GluRs is based on vertebrate proteins and has remained unchanged for over two decades. Here we report an exhaustive phylogenetic study of GluRs in metazoans. Importantly, we demonstrate that GluRs have followed different evolutionary histories in separated animal lineages. Our analysis reveals that the present organization of iGluRs into six classes does not capture the full complexity of their evolution. Instead, we propose an organization into four subfamilies and ten classes, four of which have never been previously described. Furthermore, we report a sister class to mGluR classes I-III, class IV. We show that many unreported proteins are expressed in the nervous system, and that new Epsilon receptors form functional ligand-gated ion channels. We propose an updated classification of glutamate receptors that includes our findings. Glutamate is the principal excitatory neurotransmitter in the central nervous system of animals (Fonnum, 1984; Danbolt, 2001; Pascual-Anaya and D' Aniello, 2006). It acts on two families of structurally unrelated receptors: ionotropic glutamate receptors (iGluRs), which are ligand-gated ion channels and G-protein coupled receptors (GPCRs), known as metabotropic glutamate receptors (mGluRs) (Sobolevsky et al., 2009; Conn and Pin, 1997). While fast excitatory neurotransmission is mediated by iGluRs, metabotropic receptors modulate synaptic transmission strength. iGluRs are formed by four subunits, which can be traced back to bacteria (Tikhonov and Magazanik, 2009). The current classification of iGluR subunits includes six classes: α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors, Kainate receptors, N-methyl-D-aspartate (NMDA) receptors (actually comprising three classes: NMDA1-3) and Delta receptors (Traynelis et al., 2010). iGluR subunits of the same class assemble into homo- or heterotetramers (Karakas and Furukawa, 2014; Kumar et al., 2011) and their ligand selectivity is dictated by a small number of residues located in the ligand-binding domain (Traynelis et al., 2010). Accordingly, NMDA subunits GluN1 and GluN3 as well as the Delta subunit GluD2 bind glycine and D-serine, while all subunits from the AMPA and Kainate classes bind glutamate (Traynelis et al., 2010; Kristensen et al., 2016). Metabotropic glutamate receptors are class C GPCRs and as such are formed by a single polypeptide. mGluRs also appeared before the emergence of metazoans, being present in unicellular organisms such as the amoeba Dictyostellium discoideum (Taniura et al., 2006). mGluRs are presently organized into three classes (I, II and III) and all their members respond to glutamate (Conn and Pin, 1997; Pin et al., 2003). While the phylogeny of the two families of GluRs is well characterized in vertebrates, that of the entire animal kingdom is only poorly understood. The few studies on iGluR evolution outside vertebrates concentrate on a few phyla, leaving many proteins unclassified (Greer et al., 2017; Brockie et al., 2001; Janovjak et al., 2011; Kenny and Dearden, 2013). Similarly, the vast majority of mGluRs described so far fall into the three classes described in vertebrates (Krishnan et al., 2013; Kucharski et al., 2007; Dillon et al., 2006). Although, the existence of three insect mGluRs that cluster apart from classes I-III led to propose the existence of a fourth class (Mitri et al., 2004). Here we present what to our knowledge is the most comprehensive phylogenetic study of ionotropic and metabotropic GluRs along the animal kingdom. We have favored the use of more slow-evolving species for the construction of phylogenetic trees. These species are particularly amenable to phylogenetics (Simakov et al., 2013; Simakov et al., 2015; Putnam et al., 2007) as they arguably present lower rates of molecular evolution than other organisms. Our work shows that metazoan evolution of GluRs is much more complex than previously thought. iGluRs present an overall organization into four subfamilies that were already present in the last ancestor of all metazoans. Vertebrate species only retain members of two of these subfamilies. Furthermore, we identify many lineage-specific gains, losses or expansions of GluR phylogenetic groups. Finally, we present experimental evidence showing that unreported GluRs found in the basally divergent chordate Branchiostoma lanceolatum (amphioxus) are highly expressed in the nervous system and that members of the unreported Epsilon subfamily, the most phylogenetically spread among unreported groups, can form functional ligand-gated ion channels. We have performed a systematic phylogenetic study of iGluR evolution across the animal kingdom. To increase the confidence on iGluRs evolutionary history phylogenetic trees have been generated using two independent methods (Bayesian inference and Maximum-likelihood (ML), Figure 1 and Figure 1—figure supplement 1). Our analysis indicates that the family of iGluRs experienced key duplication events that define its present organization into four previously unreported subfamilies, of which two contain the extensively studied vertebrate classes. Assuming ctenophores as the sister group to all other animals (Moroz et al., 2014; Ryan et al., 2013), our data suggest that the three major duplication events leading to this four subfamilies occurred before the divergence of current animal phyla (see Figure 2 for a summary scheme of iGluRs evolution). The first of these duplications produced the separation of the Lambda subfamily, the second lead to divergence of the NMDA subfamily and the third to the split between Epsilon and AKDF subfamilies. The Lambda subfamily is the most phylogenetically restricted, as we could only identify it in porifers. Thus, Lambda would have been lost in two occasions, in the lineage of ctenophores and in a common ancestor of placozoans, cnidarians and bilaterals. On the other hand, the Epsilon subfamily is the best represented among non-bilaterians, being present in all non-bilaterian phyla investigated. Including in porifers, although we could only identify one Epsilon in sponges, GluE_Ifa from the demosponge Ircinia fasciculata. Our data also indicate that this subfamily has been lost in multiple occasions along metazoan evolution, as we could not find it in the protostome, echinoderm or vertebrate species investigated. Interestingly, all ctenophore iGluRs identified, which have been previously reported (Alberstein et al., 2015), belong to the Epsilon subfamily. Thus, this phylum would have lost NMDA, Lambda and AKDF proteins. Contrarily, ctenophores would have experienced an important expansion of Epsilon iGluRs, as we report 17 and 10 of these proteins in the two species with genomic information available, M. leidyi and P. bachei, respectively. Although we have not identified NMDA receptors in ctenophores, porifers and placozoans our analysis indicates that this subfamily was already present in the last common ancestor of metazoans. This is because the topology of the tree shows that NMDAs appear in the phylogeny at the same level as the Epsilon subfamily, which has representatives in all non-bilateral phyla. According to our data, NMDA1s on the one hand and NMDA2s and NMDA3s on the other contain members of the cnidarian phylum. Although we have only been able to identify one member more closely related to NMDA2 and NMDA3 than NMDA1 (GluN2/3_Nve), its position in the phylogeny is very well supported by both analyses performed. This indicates that a specific duplication occurred in the ancestor of bilaterians originating NMDA2s and NMDA3s. Moreover, we have also identified a cnidarian-specific NMDA class, that we have termed NMDA-Cnidaria, this class presents representative proteins in 3 of the four species investigated. Among bilaterals we have observed conservation of all NMDA classes with the exception of NMDA2s in echinoderms, which are absent from the two species examined. Interestingly, studied cnidarian species substantially expanded their NMDA subfamily repertoire, with at least six members in Nematostella vectensis. In bilaterians the AKDF subfamily diversified into the known AMPA, Kainate and Delta classes, but also into a fourth new class that we have termed Phi. The phylogenetic spread of these classes is quite variable, as AMPA and Kainate are in all bilateral phyla investigated but Delta and Phi are more restricted. Deltas are almost completely absent from ecdysozoan species, as we could only find a single member of this class in priapulids (P. caudatus) and none in arthropods or nematodes. Similarly, Deltas are poorly represented in mollusks and, with the available data, absent in annelids. Finally, we could only identify Phi proteins in cephalochordates, hemichordates and echinoderms, indicating that this class might be lost in the lineages of protostomes and olfactores (i. e. vertebrates and urochordates). The AKDF subfamily also includes proteins from the non-bilateral phyla of porifera, placozoa and cnidarian. The exact organization of these proteins into classes is not as straightforward as for bilateral proteins. The Bayesian and ML analysis only agree in the position of 12 iGluRs from the sponge O. carmela, these would constitute the only clear class in non-bilaterals, which we have termed AKDF-Oca. Another example of a multiple lineage-specific event that occurred during animal evolution of iGluRs can be observed in the evolution of AMPA and Kainate proteins among protostomes. The general iGluRs phylogeny (Figure 1) suggests that ecdysozoan species have expanded their repertoire of Kainate subunits when compared with lophotrochozoans (e. g. mollusks, annelids), since C. teleta and L. gigantea only presents one and two genes coding for Kainate receptors, respectively. Contrarily, we found more AMPA subunits in lophotrochozoans than in ecdysozoan species. To investigate whether the two protostome lineages have alternatively expanded genes coding for AMPA or Kainate subunits we conducted a phylogenetic analysis of these two classes using eight species of ecdysozoans and seven of lophotrochozoans with well-characterized genomes (Figure 3 and Figure 3—figure supplement 1). Nematodes were left out of the analysis as they lack Kainate receptors (Brockie et al., 2001). This analysis retrieved 40 lophotrochozoan genes coding for AMPA subunits but only 15 coding for Kainates. The opposite scenario was observed in the genomes of ecdysozoan species, with 10 AMAP and 40 Kainate proteins,. Yet, among ecdysozoans the priapulid P. caudatus has two AMPA and two Kainate subunits, indicating that the expansion of Kainate receptors might be exclusive to arthropods. Overall the AMPA: Kainate ratio resulted to be around 1: 4 in ecdysozoans and 4: 1 in lophotrochozoans. All proteins from unreported groups (i. e. subfamilies and classes) present well-conserved sequences in iGluR domains, including transmembrane domains or residues involved in receptor tetramerization (Figure 1—figure supplement 2 and Figure 1—source data 1). Three-dimensional (3D) models of two Epsilon subunits from amphioxus (GluE1 and GluE7) indicate that their general fold is well preserved (Figure 1—figure supplement 3a). The only noticeable distinction in proteins from these groups is an insertion in the intracellular loop between the first and second transmembrane domains in Epsilon proteins. This insertion is particularly distinct in ctenophore iGluRs, having been termed as the cysteine-rich loop (Alberstein et al., 2015) (Figure 1—figure supplement 4). We have also identified a sequence difference among Epsilon proteins. Ctenophore iGluRs have two cysteines that form a disulfide bond at loop 1 of the ligand binding domain (Alberstein et al., 2015), which are also present in NMDA proteins. Nevertheless, this element is absent from the remaining members of the Epsilon subfamily. The ‘SYTANLAAF’ motif, essential for channel gating (Traynelis et al., 2010), is also well conserved in most sequences, in particular the second, fourth and fifth residues (Figure 1—figure supplement 2). Nevertheless, all members of the Lambda subfamily and some proteins of the Phi class present lower levels of conservation in this sequence. Whether these changes have a functional impact is something that will require further investigation. The Q/R site (Q586, residue numbering according to mature rat GluA2) and the acidic residue located four positions downstream D/E590 (Figure 1—figure supplement 4) are involved in calcium permeability and polyamine block of AMPA and Kainate receptors (Bowie and Mayer, 1995; Koh et al., 1995; Kamboj et al., 1995). Of these two positions the latter is much better conserved, especially outside ctenophores and the Lambda subfamily. We have identified an acidic residue at position 590 in 84 out of 122 iGluRs from unreported groups, including cnidarian NMDAs. Yet, only 1/3 of these proteins present a glutamine (Q) at position 586. This includes most AKDFs and Epsilon proteins from non-ctenophores, contrarily, none of the Phi subunits presents a Q586. The key ligand binding residues involved in fixing the amino acid backbone (α−amino and α−carboxyl) are Arg485 and an acidic residue at position 705 (Naur et al., 2007; Armstrong and Gouaux, 2000; Mayer, 2005; Furukawa et al., 2005; Yao et al., 2008). These two positions are well conserved in 94 of the 122 proteins from unreported groups, suggesting that their endogenous ligand is an amino acid (see Figure 1—figure supplement 3b for a 3D representation of ligand binding by GluE1 and Figure 1—figure supplement 5 for an alignment of iGluR residues involved in ligand binding). The residue changes found in the remaining 28 proteins would render them unable to bind an amino acid (Figure 1—figure supplement 5). This is are particularly common among class Phi proteins from amphioxus and in NMDA-Cnidaria. Residues involved in ligand selectivity show higher variability. These are located at positions 653 and 655, and are occupied by glycine and threonine in glutamate-binding proteins and by serine and a non-polar residue in glycine-binding iGluRs. However, a recent study of ctenophore receptors has found that position 653 can be occupied by serine or threonine in glutamate-binding iGluRs, and by an arginine in glycine-binding subunits (Alberstein et al., 2015). Based on this previous knowledge we have predicted the ligand specificities of most previously unreported receptors. The preferred ligand could be confidently predicted for 72 out of the 94 proteins with well-conserved residues involved in fixing the amino acid backbone. Interestingly, all unreported groups comprise glycine- and glutamate-specific iGluRs. Gly-specific receptors slightly outnumber those predicted to respond to glutamate (overall ratio about 3: 2). The Lambda subfamily would include three proteins specific for glutamate and one for glycine, while seven remain with an unknown selectivity. Of note, the protein predicted to bind glycine (GluL5_Oca) displays an arginine at position 653, a feature which had only been reported in ctenophores (Alberstein et al., 2015). This residue would form a salt bridge with Glu423, which is key for glycine selectivity in ctenophores (Alberstein et al., 2015). Most Epsilon and AKDF proteins would preferably bind glycine, although ctenophores present a similar number of Epsilon receptors predicted to respond to glycine or glutamate (Figure 1) (Alberstein et al., 2015). In the Phi class we also found a similar number of receptors binding glycine and glutamate. Finally, we could only predict binding specificity for two of the 9 NMDA-Cnidaria proteins, as they present many changes in the residues involved in either amino acid backbone binding or side chain recognition. Interestingly, the 22 proteins for which we could not confidently predict their ligand selectivity (Figure 1—figure supplement 5), present a limited number of residues occupying position 653 and 655, suggesting constrained evolution. Of these: (i) nine present residues with negative polarity at both positions, being candidates to bind glutamate, (ii) six present a Gly653 and a non-polar residue at position 655, and thus are candidates to bind glycine, (iii) five proteins, all from the Branchiostoma genus, present a tyrosine at position 653. A structural model of one of these receptors, GluE7 (Figure 1—figure supplement 3c), shows that a Tyr653 aromatic side chain would occupy the ligand-binding pocket, strongly suggesting that amino acid binding would be blocked. Finally, (iv) two proteins present a phenylalanine in either of the two positions and remain unclassified. We used quantitative PCR (qPCR) to investigate gene expression levels of all iGluR subunits identified in B. lanceolatum, including those from the Epsilon and Phi groups. All 24 B. lanceolatum iGluR subunits identified in silico were found expressed in amphioxus, with the exception of Grie5 (Figure 4a). Furthermore, they all showed a significantly higher expression in the nerve cord as compared to the whole body, suggesting tissue-enriched expression. While we observed low expression levels for Epsilon genes coding for subunits with a tyrosine at position 653 (Grie5-8), which according to the 3D model would block the ligand-binding pocket, the expression of Grif1-2, also presenting the same tyrosine, reach much higher levels, comparable to those of subunits from the Kainate, Delta or NMDA classes. Thus, the presence of a tyrosine at position 653 does not appear to be directly correlated with low expression levels. Amphioxus genes coding for GluE1 and GluE7 were synthesized in vitro and transiently expressed in HEK293T cells for functional studies. Wild-type GluE1 and GluE7, which are not predicted to have a canonical signal peptide by SignalP 4. 1 (Nielsen, 2017), expressed well but were not trafficked to the plasma membrane (Figure 4—figure supplement 1a–d), even though residues involved in tetramerization (Salussolia et al., 2013) are well conserved (Figure 4b). We thus synthesized new variants of these genes with the signal peptide from rat GluA2 (Figure 1—figure supplement 1cd). These constructs also expressed well (Figure 4c) and now were efficiently trafficked to the plasma membrane, as indicated by the staining observed in non-permeabilized cells (Figure 4d). Furthermore, analysis of receptor oligomerization, performed using non-denaturing gel electrophoresis and immunoblot, clearly indicates that both proteins form homotetramers in vitro (Figure 4e). We next investigated the gating properties of two Epsilon proteins from amphioxus, GluE1 and GluE7. The presence of a serine and a tryptophan at positions 653 and 704, respectively, suggested that GluE1 would bind glycine. Indeed, neither glutamate nor aspartate elicited a response in our experimental settings. Instead, glycine application was able to elicit an inward whole-cell current at a membrane potential of −60 mV (Figure 5a). Interestingly, the chemically related amino acids alanine and D-serine only generated very low responses, indicating a high selectivity of the GluE1 homotetramer for glycine. The Epsilon receptor displayed a strong inward rectification, even in the absence of added polyamines in the intracellular solution (Figure 5b, c). This behavior is characteristic of unedited AMPA and Kainate receptors displaying a glutamine (Q) and an acidic residue at positions 586 and 590, respectively (Bowie and Mayer, 1995; Koh et al., 1995; Kamboj et al., 1995) and GluE1 presents a glutamine and an aspartic acid at these positions (Figure 1—figure supplement 4). Glycine-mediated currents showed a slow rate of recovery from desensitization when compared with AMPA or Kainate mammalian receptors, requiring 20–25 seconds until a complete recovery was achieved and a full response of the same magnitude could be recorded (Figure 5d, e). Similar observations have been made with ctenophore receptors activated by glycine in which the recovery from desensitization has an unusually long time constant of 81 seconds (Alberstein et al., 2015). Finally, functional studies on receptors formed by GluE7 did not retrieve any positive results. None of the following amino acids: glutamate, aspartate, asparagine, glycine, alanine or D-serine elicited a response in our experimental system. We hypothesize that, as predicted by the 3D model, the presence of a tyrosine at position 653 renders a homomeric form of this receptor unable to function as an amino acid-gated ion channel. We next performed a phylogenetic study of metabotropic glutamate receptors (Figure 6 and Figure 6—figure supplement 1). This analysis has revealed that the three historical mGluR classes (I to III) have a sister group. Following the current nomenclature we have named this as class IV. The existence of this class had already been proposed on the bases of three insect proteins (Mitri et al., 2004). Yet, here we show that this class is actually present in all bilateral phyla, excluding vertebrates. Furthermore, we also show that class IV appeared together with classes I-III before radiation of bilateral lineages. We have identified clear orthologues to class I-IV in porifers, placozoans and cnidarians but not in ctenophores. These are organized into four classes, two from cnidarians, and one from placozoans and porifers (Figure 6). We have also identified non-bilaterian mGluRs that fall outside the above-mentioned classes. Unfortunately, the Bayesian and ML phylogenies do not agree on the exact organization of these early divergent mGluRs, except for the fact that they diverge prior to bilaterian classes. For this reason we have left these sequences unclassified. Whether these sequences belong to one, or even multiple classes that would have been lost in bilateral organisms is something that will require further investigation. Although all class IV proteins show well conserved sequences overall (Figure 6—figure supplement 2a, Figure 6—figure supplement 3 and Figure 6—source data 1), two residues critical for glutamate binding, Arg78 and Lys409, are non-conservatively replaced by non-polar or acidic residues in all class IV proteins identified (Figure 6—figure supplement 2a, residue numbering corresponds to human mGluR1). These changes are predicted to hamper glutamate binding and, indeed, functional studies of a class IV receptor from fruit fly indicated that it does not respond to this amino acid (Mitri et al., 2004). All class IV proteins would share this feature. On the other hand, residues involved in contacts with the amino acid backbone are well conserved (Figure 6—figure supplement 2a), suggesting that these proteins might bind an amino acid other than glutamate. Similarly, mGluR residues from most non-bilaterian sequences involved in binding the amino acid backbone are highly conserved. Among non-bilaterian proteins the residues involved in glutamate binding are only conserved in approximately half of the proteins from classes orthologous to I-II-III-IV. Finally, we investigated mGluRs expression in amphioxus following the same procedure described for iGluRs. All five amphioxus mGluRs showed an enriched expression in the nerve cord, including the two class IV genes. Noticeably, these two genes showed significantly higher expression levels than orthologues of vertebrate classes (Figure 6—figure supplement 2b). We have performed what to our knowledge is the most comprehensive phylogenetic study of metazoan glutamate receptors. This has revealed that their evolutionary history is much more complex than what is currently acknowledged, especially for the family of iGluRs. Our study has also revealed the existence of unreported phylogenetic groups in both ionotropic and metabotropic glutamate receptors. Importantly, our data indicate that the evolution of glutamate receptors has not occurred in an unequivocal incremental manner only in those clades with more elaborated neural systems, but it has rather followed an scattered lineage-specific evolutionary history. This means that certain lineages have experienced the gain, loss, expansion or reduction of specific phylogenetic groups. Our phylogenetic analysis indicates that the family of iGluRs is actually divided into four unreported subfamilies that we have termed Lambda, Epsilon, NMDA and AKDF. Interestingly, this general organization was already present in the last common ancestor of all metazoans and later duplications within NMDA and AKDF subfamilies resulted in the formation of well-known iGluR classes. The other two subfamilies are absent from the majority of model species used in neuroscience research. The NMDA subfamily diversified into classes NMDA1-3 but also into the NMDA2/3 and NMDA-Cnidaria. Similarly, the AKDF subfamily diversified into the AMPA, Kainate and Delta classes, but also into the previously unreported Phi class. We have also identified and AKDF class exclusive to porifers, represented by sequences form O. carmela. Most well-studied iGluR classes are the result of duplications in ancestors of current bilateral species, >650 million years ago (mya) (Kumar et al., 2017), only class NMDA1 originated earlier, as cnidarians present members within this class. The Epsilon subfamily, which includes all iGluRs from ctenophores, is the only subfamily present in all non-bilateral phyla investigated, including sponges. It is thus the subfamily presenting a larger phylogenetic spread, as it is also present in hemichordates and in non-vertebrate chordates. On the other hand, the unreported Phi class shows a more restricted phylogenetic spread, as it is present only in three deuterostome phyla. Moreover, Lambda proteins seem restricted to Porifers, which constitutes an interesting evolutionary case due to maintenance of a glutamate receptor family in a phylum without nervous system. The phylogenetic analysis of metabotropic glutamate receptors has allowed us to unambiguously establish the existence of a sister group to the well-known classes I, II and III. Following the present nomenclature we have named this as class IV. This class had been previously proposed based on the identification of three insect mGluRs that did not cluster with members of known classes (Mitri et al., 2004). Here we show that class IV is not restricted to insects, but is actually present in all bilaterian phyla investigated, with the exception of vertebrates where this class has been lost. Interestingly, as it occurs for most well-known iGluR classes, mGluR classes I-IV appeared simultaneously in the ancestor of bilaterals. Our phylogenetic analysis also indicates that the non-bilateral phyla of cnidarians, placozoans and porifers present clear orthologues to classes I-IV, which are organized into four classes, while we failed to find any in the early-branching ctenophores. Finally, we were unable to confidently classify many non-bilateral mGluRs, which might constitute one or more classes. We have identified many examples of lineage-specific evolutionary events. These would antagonize with a model in which species with less elaborated nervous systems would present GluR families with lower complexity. The most noticeable examples are: (i) the absence of all subfamilies but Epsilon in analyzed ctenophores, (ii) the loss of Delta receptors from arthropods, nematodes and annelid species investigated, (iii) the loss of the Epsilon subfamily in vertebrates, echinoderms and protostomes, (iv) the loss of the Phi class in vertebrates and studied protostomes, (v) the specific expansion of Kainate receptors in arthropods, which contrasts with the expansion of AMPA receptors in its sister lineages of mollusks and annelids, (vi) the large expansion of the Epsilon subfamily in ctenophores, placozoans and cephalochordates and, finally (vii) the loss of mGluR class IV in vertebrates. Along the same line, it is interesting to note that amphioxus (B. belcheri and B. lanceolatum), with a simple nervous system, have over 20 genes encoding iGluRs, while mammals have 18. Other non-vertebrate species also present large numbers of iGluRs, including the 19 iGluRs identified in the sponge O. carmela or the 17 present in the ctenophore M. leidyi, to mention a few. Similarly, the cnidarian A. digitifera and the ctenophore M. leidyi have seven mGluRs each, while the placozoan T. adhaerens presents eleven, three more than the eight mGluRs found in the human genome. The large number of GluRs found in many non-vertebrate animals suggests that there has been an evolutionary trend to increase their number in many metazoan lineages. Our experimental results suggest that unreported receptors would play a role in the nervous system, as Epsilon, Phi and mGluR class IV genes are highly expressed in the nerve cord of amphioxus. Nevertheless, whether all these proteins are expressed at the synapse and act as neurotransmitter receptors is an issue that will require further investigation. Their presence in other tissues, such as sensory organs, cannot be ruled out. Those receptors showing more divergent sequences, particularly in residues involved in ligand binding, might respond to other molecules. For instance, they could behave as chemoreceptors, as it is the case of antennal receptors found in insects (Croset et al., 2010; Benton et al., 2009). Proteins from all unreported groups generally present a good conservation of residues involved in binding the amino acid backbone, indicating that their ligand would be an amino acid or a closely related molecule. Interestingly, we could identify proteins predicted to bind either glycine or glutamate in all unreported iGluR subfamilies and classes. If our functional predictions are correct, the ability to recognize one or the other amino acid would have emerged repeatedly in all unreported iGluR phylogenetic groups. Unexpectedly, the nature of the residues conferring amino acid specificity indicates that only a minority of proteins from unreported GluR groups would respond to glutamate. Sequence analysis and structural considerations strongly suggest that class IV mGluRs will not bind glutamate and that among non-bilateral mGluRs only a minority, belonging to classes orthologous to I-II-III-IV, are predicted to bind to this neurotransmitter. Similarly, among unreported iGluR groups, the number of proteins binding glycine outnumbers those binding glutamate. Interestingly, we report a glycine-binding poriferan protein (GluL5_Oca) with a structural feature that had only been reported in ctenophores (Alberstein et al., 2015). This is an Arg653 that through establishing a salt bridge with Glu423 confers glycine specificity (Alberstein et al., 2015). We thus report that this structural element is not exclusive to ctenophores. We have also identified iGluR subunits with important changes in critical ligand binding residues, indicating that they might have evolved new biological functions, for example, response to other, as yet unidentified small molecules. The activation of Epsilon receptors by glycine has been experimentally corroborated by electrophysiological analysis of homotetrameric receptors composed by GluE7 from M. leidy (Alberstein et al., 2015) and GluE1 from amphioxus (this study). In our hands the amphioxus receptor showed a very high selectivity for glycine, since ion currents could not be elicited by chemically related amino acids such as serine or alanine. Glycine-binding Epsilon subunits from phyla other than ctenophores present structural features similar to those from glycine-binding iGluRs in vertebrates. The greater number of glycine receptors found in non-vertebrate species could be related to the higher abundance of this amino acid in their nerve cord as compared with the mammalian brain (Pascual-Anaya and D' Aniello, 2006). Altogether, our phylogenetic analysis and experimental findings have uncovered the complex evolution of glutamate receptors within the metazoan kingdom. Our data indicate that the classification of iGluRs is not restricted to the six classes currently recognized. Instead, iGluRs are organized into four subfamilies: Lambda, Epsilon, NMDA and AKDF and ten classes with varying phylogenetic spread. With the data available, the NMDA subfamily is organized into classes NMDA1, NMDA 2, NMDA3, NMDA-Cnidaria and NMDA2/3, while subfamily AKDF contains classes AMPA, Kainate, Delta, Phi and AKDF-Oca. Both NMDA2/3 and AKDF-Oca are represented by sequences from only one species, further sequencing of non-bilateral species will be required to fully demonstrate their existence. Furthermore, the evolution of mGluRs has generated a sister group to classes I, II and III, class IV. We have also identified classes of non-bilaterian mGluRs orthologous to I-II-III-IV. We propose that the classification of these two families of GluRs, key to the physiology of the nervous system, has to be updated to include our findings. Phylogenetic analysis were performed with sequences from at least two species from each of the following metazoan phyla: Porifera, Ctenophora, Placozoa, Cnidaria, Lophotrochozoa, Ecdysozoa, Hemichordata, Chordata and Vertebrata, with the exception of placozoans for which only one species is available. When possible, we chose slowly evolving species. The complete lists of species used for iGluR phylogenies are given in Figure 1—source data 2. Species used in the phylogeny of metabotropic glutamate receptors are listed in Figure 6—source data 2. Sponge sequences were taken from (Riesgo et al., 2014), B. lanceolatum sequences were retrieved from unpublished genomic and transcriptomic databases (access was kindly provided by the Mediterranean Amphioxus Genome Consortium), A. digitifera and P. flava sequences were obtained from the Marine Genomics Unit (Simakov et al., 2015; Shinzato et al., 2011) and P. bachei sequences from NeuroBase (Moroz et al., 2014). GluR sequences were identified using homology-based searches in a two-tier approach. Mouse glutamate receptors were used as search queries (iGluRs: Gria1-4; Grik1-5; Grid1-2, Grin1, Grin2A-D and Grin-3A-B; mGluRs: mGluR1-8). In a first search GluR homologs were identified using the BLASTP tool (Altschul et al., 1990) with default parameters. Subject sequences with an E-value below 0. 05 were selected as candidate homologs. These were re-blasted against the NCBI database of ‘non-redundant protein sequences’ using the same BLAST tool. If the first hit obtained in the reciprocal BLAST was a glutamate receptor the sequence was included in the phylogenetic analysis. In a second stage the same mouse sequences were used to perform TBLASTN searches against genomic and, when available, transcriptomic databases. Subject sequences not identified in the first tear and having an E-value below 0. 05 were selected as candidate homologs. These were re-blasted using BLASTX against the NCBI ‘non-redundant protein sequences’ database. Finally, if the first hit of this search was a glutamate receptor the sequence was also included in the phylogenetic analysis. Identified iGluR sequences in which less than four residues of the SYTANLAAF motif (Traynelis et al., 2010) were conserved were not considered for the final phylogenetic analysis. mGluR sequences lacking two or more of the seven transmembrane regions were also discarded. The complete reference lists of all iGluRs used in the final phylogeny are given in files Figure 1—source data 2. The reference list of metabotropic glutamate receptors is presented in Figure 6—source data 2. The alignments used for the phylogenetic analysis of iGluRs, mGluRs and AMPAs and Kainates from protostomes are provided in Figure 1—source data 3, Figure 3—source data 1 and Figure 6—source data 3. The iGluR tree was constructed with 224 sequences identified in 26 non-vertebrate species (Figure 1—source data 2). The tree also included 18 iGluR sequences from vertebrates and two iGluR proteins from A. thaliana, used as an outgroup (Chiu et al., 2002). The phylogenetic analysis of AMPA and Kainate classes in protostomes was inferred using 110 sequences from 15 protostome species (Figure 3—source data 2) and 37 sequences from deuterostomes, of which 4 GluN1 proteins were used as an outgroup. The mGluR tree was constructed with 149 proteins from 29 non-vertebrate species, 38 mGluRs from vertebrate species and 10 sequences from vertebrate metabotropic GABA receptors, used as an outgroup (Figure 6—source data 2). Protein sequences were aligned with the MUSCLE algorithm (Edgar, 2004), included in the software package MEGA6 (Tamura et al., 2013) with default parameters. ProtTest v3. 4. 2 was used to establish the best evolutionary model (Darriba et al., 2011). Trees were constructed using MrBayes v3. 2. 6 (Ronquist et al., 2012) for Bayesian inference and IQ-TREE (Nguyen et al., 2015) for Maximum-likelihood analysis. For Bayesian inference phylogenies were stopped when standard deviation was below 0. 01 and its value was fluctuating but not decreasing. Markov chain Monte Carlo (MCMC) was used to approximate the posterior probability of the Bayesian trees. Bayesian analyses included two independent MCMC runs, each using four parallel chains composed of three heated and one cold chain. Twenty-five % of initial trees were discarded as burn-in. Convergence was assessed when potential scale reduction factor (PSRF) value was between 1. 002 and 1. 000. In Maximum-likelihood analysis the starting tree was estimated using a neighbor-joining method and branch support was obtained after 1000 iterations of ultrafast bootstrapping (Hoang et al., 2018). Gene/protein names were given based on their position in the tree. Phylogenetic trees were rendered using FigTree (http: //tree. bio. ed. ac. uk/software/figtree/). Phylogenetic calculations were performed at the IBB - UAB heterogeneous computer cluster ‘Celler’ and at the CIPRES science gateway (RRID: SCR_008439) (Miller et al., 2010). Branchiostoma lanceolatum adults were collected in the bay of Argelès-sur-Mer, France (latitude 42° 32’ 53’ N and longitude 3° 03’ 27’ E) with a specific permission delivered by the Prefect of Region Provence Alpes Côte d’Azur. B. lanceolatum is not a protected species. Animals were kept in tanks with seawater at 17°C under natural photoperiod. Adult amphioxus (B. lanceolatum) were anesthetized in 0. 1% diethyl pyrocarbonate (DEPC; Sigma, D5758) PBS buffer. Animals were sacrificed by cutting the most anterior part of the body. The nerve chord was surgically extracted from the animal while submerged in DEPC-PBS using a magnifying glass. Individual nerve chords were snap frozen in liquid nitrogen and stored at −80°C until use. RNA was extracted from whole animals or from dissected nerve chords. Ten nerve chords were used for each RNA extraction, so that biological variability between individuals could be normalized. The tissue was homogenized in 1 mL of TRI Reagent (Sigma, T9424) using a Polytron homogenizer. Homogenates were transferred into an Eppendorf tube and incubated 5 min at room temperature (RT) before adding 100 µL of 1-bromo-3-cloropropane. Tubes were vigorously mixed by vortexing for 10–15 s, incubated 15 min at RT and centrifuged at 13000 rpm for 15 min at 4°C. RNA was precipitated from the aqueous phase with 500 µL of isopropanol and 20 µg of glycogen. Tubes were frozen for 1 hr at −80°C and then thawed, incubated at RT for 10 min and centrifuged at 13000 rpm for 10 min at 4°C. The RNA pellet was washed twice with 500 µL of 75% ethanol and air-dried. cDNA was synthesized from 0. 5 µg of total RNA. One µL of Oligo (dT) 15 (Promega), 1 µL of 10 mM dNTP mix (Biotools), RNA and DEPC distilled water were mixed in a PCR tube to a final volume of 14 µL. This mix was incubated at 65°C for 5 min in a T100 Thermal Cycler (BioRad). After cooling tubes on ice for 1 min, we added 4 µL of First Strand 5x buffer, 1 µL of 0. 1 M DTT and 1 µL of SuperScript III (Invitrogen). Tubes were placed in a T100 Thermal Cycler (BioRad) with the following program: 60 min at 50°C, 15 min at 70°C. RNA expression levels were determined using qPCR and the GAPDH gene used as a reference. Primers used for qPCR analysis of iGluRs are in Figure 4—figure supplement 2 and those used for mGluR qPCR in Figure 6—figure supplement 4. qPCR data for iGluRs and mGluRs are given in Figure 4—source data 1 and Figure 6—source data 4, respectively. cDNA from nerve chord and whole body samples was diluted 1: 10 for the glutamate receptor gene reactions, and 1: 100 for the reference gene reaction. For each gene 2. 5 µL of diluted cDNA were added to 5 μL of iTaq Universal SYBR Green Supermix (Bio-Rad), along with 0. 5 µL of each primer and 1. 5 µL of RNase free water. qPCR was run in a C1000 Touch thermocycler combined with the optic module CFX96. Three technical replicates were performed for all genes analyzed. Primer pairs were designed to detect the expression levels of each glutamate receptor (Figure 4—figure supplement 2 and Figure 6—figure supplement 4). B. belcheri glutamate receptor sequences were aligned with the genomic sequence of B. lanceolatum, and high identity fragments were used to design primers. All primers were 20–25 base pair long, had GC content over 40–45% and a Tm between 60–65°C. Primers were designed to obtain amplicons between 140–270 base pairs. Values of normalized expression were statistically analyzed using GraphPad Prism5. No outliers were identified and no data points were excluded. Comparisons between whole body and nerve chord expression levels were done with Student’s T-Test for unpaired samples or the Welch variant of the Student’s T-Test for samples with different variance. For multiple comparisons between the expression levels of genes belonging to the same class one-way ANOVA analysis was performed using Tukey’s Post-Hoc test. Grie1 and Grie7 genes were selected for transient expression in the mammalian cell line HEK293T. We prepared two constructs for each gene. We first introduced an immuno-tag in the N-terminus before the first element of secondary structure. For Grie1 we used the c-Myc tag, which was placed after residue 39, and for Grie7 we used the hemagglutinin (HA) tag introduced after residue 10 of the wild-type sequence. The second set of constructs prepared substituted the wild type N-terminal sequence for the signal peptide from rat GluA2 while maintaining the immuno-tags (Figure 4—figure supplement 1). Codon-optimized genes for expression in human cells were synthesized and cloned into pICherryNeo (Addgene, 52119) and pIRES2_EGFP (Addgene 6029–1) by the Invitrogen GeneArt Gene Synthesis service. All expression experiments were done with a mycoplasma-free HEK293T cell line kindly provided by Prof. F. Ciruela (Universitat de Barcelona) and purchased from the American Type Culture Collection (ATCC, CRL-3216, RRID: CVCL_0063). The ATCC has confirmed the identity of HEK293T by STR profiling (STR Profile; CSF1PO: 11,12; D13S317: 12,14; D16S539: 9,13; D5S818: 8,9; D7S820: 11; TH01: 7,9. 3; TPOX: 11; vWA: 16,19; Amelogenin: X). After the purchase of the cell line, mycoplasma tests are performed in the laboratory on every new defrosted aliquot. The kit used for mycoplasma detection is PlasmoTest (Invivogen, code: rep-pt1). HEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% FBS and 1% Antibotic-Antimycotic (Gibco) in a humidified incubator at 5% CO2 air and 37°C. The day before transfection, cells were plated onto poly-D-lysine coated coverslips in 6-well plates, to reach 60–80% confluence. HEK293T cells were transiently transfected with the following plasmids: empty pIRES2-EGFP, pIRES2-EGFP containing the Grie7_Bbe gene, empty pICherryNeo and pICherryNeo containing Grie1_Bla. Cells were transfected using 3 μg of polyethylenimine and 1 μg of plasmid DNA for each ml of non-supplemented DMEM. Cells were incubated 4–5 hr with transfection medium without supplementation, which was then removed and replaced by supplemented medium. Twenty-four hours after transfection the medium was removed and cells were washed 3 times with PBS. For surface receptor staining, cells were blocked in 2% BSA in PBS for 10 min at 37°C, and incubated for 25 min at 37°C with primary antibodies against HA (Covance, MMS-101P, RRID: AB_291259), c-Myc (Cell Signalling, 2272S, RRID: AB_10692100) or GluA2 (Millipore, MAB397, RRID: AB_2113875). HA and GluA2 antibodies were diluted 1: 200 and c-Myc 1: 100 in DMEM without supplementation. Cells were washed 3 times with PBS, fixed in 4% paraformaldehyde (PFA) for 15 min at RT, rinsed in PBS and incubated 1 hr at 37°C with secondary antibodies Alexa Fluor 555 donkey anti-mouse IgG (H + L) (A-31570, Invitrogen, RRID: AB_2536180) and Alexa Fluor 647 goat anti-rabbit IgG (H + L) highly cross-adsorbed (Life Technologies, A-21245, RRID: AB_2535813), diluted 1: 1000 and 1: 500 in PBS, respectively. Finally, coverslips were washed and mounted onto slides with Fluoroshield with DAPI (Sigma-Aldrich, F6057). For intracellular labeling cells were first fixed in 4% PFA for 15 min at RT, permeabilized with 0. 2% Triton X-100 in PBS for 10 min, and finally blocked with PBS containing 2% BSA and 0. 2% Triton X-100 for 20 min. Primary antibodies against HA (Covance, MMS-101P, RRID: AB_291259) and GluA2 (Millipore, MAB397, RRID: AB_2113875) were diluted 1: 1000 and c-Myc (Cell Signalling, 2272S, RRID: AB_10692100) antibody was prepared at 1: 100 in PBS. Incubation lasted 25 min at 37°C. Secondary antibody incubations and coverslip mounting were done in the same way as for non-permeabilized cells. Cells were examined using a confocal laser-scanning microscope (Zeiss LSM 700) with a 63x oil objective. HEK293T cells were grown in 6-well plates as described previously and transfected with plasmids expressing amphioxus GluE1, GluE7 or GluA2. Twenty-four hours after transfection cells were rinsed with PBS and the content of 4 wells was resuspended in solubilization buffer (PBS containing 2% N-dodecyl-α-maltopyranoside (DDM; D310HA, Anatrace) and the protease inhibitors mix cOmplete EDTA-free Protease Inhibitor Cocktail, Roche). Cell lysates were homogenized in a Dounce homogenizer in ice with 20 strokes and kept under orbital agitation for 1 hr at 4°C. Lysates were centrifuged at 89000xg in a Beckman TLA120. 2 rotor for 40 min at 4°C. The supernatant containing solubilized membrane proteins was recovered in a new tube and stored at −20°C until used. For native gel electrophoresis proteins were resolved in a Mini-PROTEAN TGX Gel 4–20% (Bio-Rad). Samples were mixed with Native Sample Buffer (Bio-Rad) and run along with HiMark Pre-Stained Protein Standard (Life Technologies). Electrophoresis was performed in ice at a constant voltage of 100 V for 180 min. Gels were transferred at constant current (35 mA) to polyvinylidene fluoride (PVDF) membranes overnight (16–18 hr) at 4°C. After transfer, membranes were blocked for 1 hr with Odyssey Blocking Buffer (Li-cor) in TBS, and incubated overnight at 4°C with primary antibodies anti-HA (Covance, MMS-101P, RRID: AB_291259), anti-c-Myc (Cell Signaling, 2272S, RRID: AB_10692100) or anti-GluA2 (Millipore, MAB397, RRID: AB_2113875) diluted 1: 1000 in TTBS (TBS containing 0. 05% Tween-20). After three 15 min washes in TTBS, membranes were incubated with donkey anti-mouse (Li-cor, 926–32212, RRID: AB_621847) and donkey anti-rabbit (Li-cor, 926–68073, RRID: AB_10954442) diluted 1: 7500 in TTBS for 1 hr. Blots were analyzed in an Odyssey scanner (Li-cor). For denaturing gel electrophoresis (SDS-PAGE) protein lysates were denatured by adding loading sample buffer 10x (500 mM Tris-HCl pH 7. 4,20% SDS, 10% β-mercaptoethanol, 10% glycerol and 0. 04% bromophenol blue), and incubated for 5 min at 95°C. Protein lysates were loaded in a 10% SDS- polyacrylamide gel and separated at a constant current (25 mA). Gels were transferred at a constant voltage of 100 V for 90 min in ice. Membranes were blocked for 1 hr with Odyssey Blocking Buffer in TBS, and incubated overnight at 4°C with the same primary antibodies at the same dilution as for native gels in TBS containing 0. 1% Tween 20. After three 15 min washes in TTBS, membranes were incubated with secondary antibodies as above. Blots were analyzed in an Odyssey scanner. Models for full-length GluE1 and GluE7 were generated with RaptorX (Källberg et al., 2012) based on deposited three-dimensional crystal structures of the full-length AMPA-subtype ionotropic glutamate receptor from Rattus norvegicus, GluA2, bound to competitive antagonists (PDB codes 4U4G (Yelshanskaya et al., 2014) and 3KG2 (Sobolevsky et al., 2009), respectively). Models of their respective ligand binding domains were generated with SWISS-MODEL (Biasini et al., 2014) using the atomic-resolution crystal structure of the rat GluA2 LBD bound to glutamate as template (PDB code 4YU0). Model quality was assessed with MolProbity (http: //molprobity. biochem. duke. edu/, RRID: SCR_014226). MolProbity scores for all models are given in Figure 1—source data 4. Models were inspected with MIFit (Smith, 2010) and figures were prepared with PyMOL (www. pymol. org). Cells were visualized with an inverted epifluorescence microscope (AxioVert A. 1, Zeiss) and were constantly perfused at 22–25°C with an extracellular solution containing (in mM): 145 NaCl, 2. 5 KCl, 2 CaCl2,1 MgCl2,10 HEPES and 10 glucose (pH = 7. 42 with NaOH; 305 mOsm/Kg). Microelectrodes were filled with an intracellular solution containing (in mM): 145 CsCl, 2. 5 NaCl, 1 Cs-EGTA, 4 MgATP, 10 HEPES (pH = 7. 2 with CsOH; 295 mOsm/Kg). Electrodes were fabricated from borosilicate glass (1. 5 mm o. d., 1. 16 i. d., Harvard Apparatus) pulled with a P-97 horizontal puller (Sutter Instruments) and polished with a forge (MF-830, Narishige) to a final resistance of 2–4 MΩ. Currents were recorded with an Axopatch 200B amplifier filtered at 1 KHz and digitized at 5 KHz using Digidata 1440A interface with pClamp 10 software (Molecular Devices Corporation). Whole-cell macroscopic currents were recorded from isolated or coupled pairs of mCherry or EGFP positive HEK293T cells. Rapid application (<1 ms exchange) of agonists (500 ms pulses) at a membrane potential of −60 mV was achieved by means of a theta-barrel tool (1. 5 mm o. d. ; Sutter Instruments) coupled to a piezoelectric translator (P-601. 30; Physik Instrumente). One barrel contained extracellular solution diluted to 96% with H2O and the other barrel contained 10 mM of the amino acid solution. For measuring current-voltage relationships, 500 ms agonist jumps were applied at different membrane voltages (−80 mV to +80 mV in 20 mV steps) and peak currents were fitted to a 5th order polynomial function. To study recovery from desensitization, a two-pulse protocol (500 ms each) was used in which a first pulse was applied followed by a second pulse at different time intervals (from 2. 5 s to 25 s). The paired pulses were separated 30–60 s to allow full recovery from desensitization. To estimate the percentage of recovery, the magnitude of peak current at the second pulse (P2) was compared with the first one (P1). Electrophysiological recordings were analyzed using IGOR Pro (Wavemetrics Inc.) with NeuroMatic (Jason Rothman, UCL, RRID: SCR_004186). | Title: Metazoan evolution of glutamate receptors reveals unreported phylogenetic groups and divergent lineage-specific events Summary: Nerve cells or neurons communicate with each other by releasing specific molecules in the gap between them, the synapses. The sending neuron passes on messages through packets of chemicals called neurotransmitters, which are picked up by the receiving cell with the help of receptors on its surface. Neurons use different neurotransmitters to send different messages, but one of the most common ones is glutamate. There are two families of glutamate receptors: ionotropic receptors, which can open or close ion channels in response to neurotransmitters and control the transmission of a signal, and metabotropic receptors, which are linked to a specific protein and control the strength of signal. Our understanding of these two receptor families comes from animals with backbones, known as vertebrates. But the receptors themselves are ancient. We can trace the first family back as far as bacteria and the second back to single-celled organisms like amoebas. Vertebrates have six classes of ionotropic and three classes of metabotropic glutamate receptor. But other multi-celled animals also have these receptors, so this picture may not be complete. Here, Ramos-Vicente et al. mapped all major lineages of animals to reveal the evolutionary history of these receptors to find out if the receptor families became more complicated as brain power increased. The results showed that the glutamate receptors found in vertebrates are only a fraction of all the types that exist. In fact, before present-day animal groups emerged, the part of the genome that holds the ionotropic receptor genes duplicated three times. This formed four receptor subfamilies, and our ancestors had all of them. Across the animal kingdom, there are ten, not six, classes of ionotropic receptors and there is an extra class of metabotropic receptors. But only two subfamilies of ionotropic and three out of four metabotropic receptor classes are still present in vertebrates today. The current classification of glutamate receptors centers around vertebrates, ignoring other animals. But this new data could change that. A better knowledge of these new receptors could aid neuroscientists in better understanding the nervous system. And, using this technique to study other families of proteins could reveal more missing links in evolution. | 14,478 | 522 | lay_elife | en |
Summarize: A Love-Scene Poor Tom bore his severe pain heroically, and was resolute in not "telling" of Mr. Poulter more than was unavoidable; the five-shilling piece remained a secret even to Maggie. But there was a terrible dread weighing on his mind, so terrible that he dared not even ask the question which might bring the fatal "yes"; he dared not ask the surgeon or Mr. Stelling, "Shall I be lame, Sir?" He mastered himself so as not to cry out at the pain; but when his foot had been dressed, and he was left alone with Maggie seated by his bedside, the children sobbed together, with their heads laid on the same pillow. Tom was thinking of himself walking about on crutches, like the wheelwright's son; and Maggie, who did not guess what was in his mind, sobbed for company. It had not occurred to the surgeon or to Mr. Stelling to anticipate this dread in Tom's mind, and to reassure him by hopeful words. But Philip watched the surgeon out of the house, and waylaid Mr. Stelling to ask the very question that Tom had not dared to ask for himself. "I beg your pardon, sir,--but does Mr. Askern say Tulliver will be lame?" "Oh, no; oh, no," said Mr. Stelling, "not permanently; only for a little while." "Did he tell Tulliver so, sir, do you think?" "No; nothing was said to him on the subject." "Then may I go and tell him, sir?" "Yes, to be sure; now you mention it, I dare say he may be troubling about that. Go to his bedroom, but be very quiet at present." It had been Philip's first thought when he heard of the accident,--"Will Tulliver be lame? It will be very hard for him if he is"; and Tom's hitherto unforgiven offences were washed out by that pity. Philip felt that they were no longer in a state of repulsion, but were being drawn into a common current of suffering and sad privation. His imagination did not dwell on the outward calamity and its future effect on Tom's life, but it made vividly present to him the probable state of Tom's feeling. Philip had only lived fourteen years, but those years had, most of them, been steeped in the sense of a lot irremediably hard. "Mr. Askern says you'll soon be all right again, Tulliver, did you know?" he said rather timidly, as he stepped gently up to Tom's bed. "I've just been to ask Mr. Stelling, and he says you'll walk as well as ever again by-and-day." Tom looked up with that momentary stopping of the breath which comes with a sudden joy; then he gave a long sigh, and turned his blue-gray eyes straight on Philip's face, as he had not done for a fortnight or more. As for Maggie, this intimation of a possibility she had not thought of before affected her as a new trouble; the bare idea of Tom's being always lame overpowered the assurance that such a misfortune was not likely to befall him, and she clung to him and cried afresh. "Don't be a little silly, Magsie," said Tom, tenderly, feeling very brave now. "I shall soon get well." "Good-by, Tulliver," said Philip, putting out his small, delicate hand, which Tom clasped immediately with his more substantial fingers. "I say," said Tom, "ask Mr. Stelling to let you come and sit with me sometimes, till I get up again, Wakem; and tell me about Robert Bruce, you know." After that, Philip spent all his time out of school-hours with Tom and Maggie. Tom liked to hear fighting stories as much as ever, but he insisted strongly on the fact that those great fighters who did so many wonderful things and came off unhurt, wore excellent armor from head to foot, which made fighting easy work, he considered. He should not have hurt his foot if he had had an iron shoe on. He listened with great interest to a new story of Philip's about a man who had a very bad wound in his foot, and cried out so dreadfully with the pain that his friends could bear with him no longer, but put him ashore on a desert island, with nothing but some wonderful poisoned arrows to kill animals with for food. "I didn't roar out a bit, you know," Tom said, "and I dare say my foot was as bad as his. It's cowardly to roar." But Maggie would have it that when anything hurt you very much, it was quite permissible to cry out, and it was cruel of people not to bear it. She wanted to know if Philoctetes had a sister, and why _she_ didn't go with him on the desert island and take care of him. One day, soon after Philip had told this story, he and Maggie were in the study alone together while Tom's foot was being dressed. Philip was at his books, and Maggie, after sauntering idly round the room, not caring to do anything in particular, because she would soon go to Tom again, went and leaned on the table near Philip to see what he was doing, for they were quite old friends now, and perfectly at home with each other. "What are you reading about in Greek?" she said. "It's poetry, I can see that, because the lines are so short." "It's about Philoctetes, the lame man I was telling you of yesterday," he answered, resting his head on his hand, and looking at her as if he were not at all sorry to be interrupted. Maggie, in her absent way, continued to lean forward, resting on her arms and moving her feet about, while her dark eyes got more and more fixed and vacant, as if she had quite forgotten Philip and his book. "Maggie," said Philip, after a minute or two, still leaning on his elbow and looking at her, "if you had had a brother like me, do you think you should have loved him as well as Tom?" Maggie started a little on being roused from her reverie, and said, "What?" Philip repeated his question. "Oh, yes, better," she answered immediately. "No, not better; because I don't think I _could_ love you better than Tom. But I should be so sorry,--_so sorry_ for you." Philip colored; he had meant to imply, would she love him as well in spite of his deformity, and yet when she alluded to it so plainly, he winced under her pity. Maggie, young as she was, felt her mistake. Hitherto she had instinctively behaved as if she were quite unconscious of Philip's deformity; her own keen sensitiveness and experience under family criticism sufficed to teach her this as well as if she had been directed by the most finished breeding. "But you are so very clever, Philip, and you can play and sing," she added quickly. "I wish you _were_ my brother. I'm very fond of you. And you would stay at home with me when Tom went out, and you would teach me everything; wouldn't you,--Greek and everything?" "But you'll go away soon, and go to school, Maggie," said Philip, "and then you'll forget all about me, and not care for me any more. And then I shall see you when you're grown up, and you'll hardly take any notice of me." "Oh, no, I sha'n't forget you, I'm sure," said Maggie, shaking her head very seriously. "I never forget anything, and I think about everybody when I'm away from them. I think about poor Yap; he's got a lump in his throat, and Luke says he'll die. Only don't you tell Tom. because it will vex him so. You never saw Yap; he's a queer little dog,--nobody cares about him but Tom and me." "Do you care as much about me as you do about Yap, Maggie?" said Philip, smiling rather sadly. "Oh, yes, I should think so," said Maggie, laughing. "I'm very fond of _you_, Maggie; I shall never forget _you_," said Philip, "and when I'm very unhappy, I shall always think of you, and wish I had a sister with dark eyes, just like yours." "Why do you like my eyes?" said Maggie, well pleased. She had never heard any one but her father speak of her eyes as if they had merit. "I don't know," said Philip. "They're not like any other eyes. They seem trying to speak,--trying to speak kindly. I don't like other people to look at me much, but I like you to look at me, Maggie." "Why, I think you're fonder of me than Tom is," said Maggie, rather sorrowfully. Then, wondering how she could convince Philip that she could like him just as well, although he was crooked, she said: "Should you like me to kiss you, as I do Tom? I will, if you like." "Yes, very much; nobody kisses me." Maggie put her arm round his neck and kissed him quite earnestly. "There now," she said, "I shall always remember you, and kiss you when I see you again, if it's ever so long. But I'll go now, because I think Mr. Askern's done with Tom's foot." When their father came the second time, Maggie said to him, "Oh, father, Philip Wakem is so very good to Tom; he is such a clever boy, and I _do_ love him. And you love him too, Tom, don't you? _Say_ you love him," she added entreatingly. Tom colored a little as he looked at his father, and said: "I sha'n't be friends with him when I leave school, father; but we've made it up now, since my foot has been bad, and he's taught me to play at draughts, and I can beat him." "Well, well," said Mr. Tulliver, "if he's good to you, try and make him amends, and be good to _him_. He's a poor crooked creature, and takes after his dead mother. But don't you be getting too thick with him; he's got his father's blood in him too. Ay, ay, the gray colt may chance to kick like his black sire." The jarring natures of the two boys effected what Mr. Tulliver's admonition alone might have failed to effect; in spite of Philip's new kindness, and Tom's answering regard in this time of his trouble, they never became close friends. When Maggie was gone, and when Tom by-and-by began to walk about as usual, the friendly warmth that had been kindled by pity and gratitude died out by degrees, and left them in their old relation to each other. Philip was often peevish and contemptuous; and Tom's more specific and kindly impressions gradually melted into the old background of suspicion and dislike toward him as a queer fellow, a humpback, and the son of a rogue. If boys and men are to be welded together in the glow of transient feeling, they must be made of metal that will mix, else they inevitably fall asunder when the heat dies out. | Summary: Tom is terribly worried that he will be lame, or have a limp, for the rest of his life. Mr. Stelling doesn't think to reassure Tom, but luckily Philip asks about it and goes to tell Tom the good news: he won't have a permanent injury. Tom and Philip reconcile and Philip hangs out with Tom and Maggie, telling them fun stories and Greek myths. A few days later Maggie and Philip are alone in the school room. Philip asks Maggie if she could love a brother like him and Maggie says yes, but that she'd love Tom best still. But she'd feel sorry for a deformed brother. This makes Philip uncomfortable and Maggie quickly assures Philip that she thinks he's very smart and likes him a lot. Maggie also assures Philip she won't forget him when she goes away and Philip says he'll always remember her. He wishes he had a sister like her. Philip tells Maggie that he likes her eyes and Maggie is surprised to note that Philip seems to like her better than Tom. Maggie gives Philip a kiss on the cheek. When Mr. Tulliver comes to fetch Maggie, she praises Philip to him. Mr. Tulliver tells Tom not to get overly friendly with Philip, since he is a Wakem. Once Tom gets better, the two boys grow apart again due to their differing personalities. | 2,649 | 307 | booksum | en |
Write a title and summarize: Phototrophic organisms such as cyanobacteria utilize the sun’s energy to convert atmospheric carbon dioxide into organic carbon, resulting in diurnal variations in the cell’s metabolism. Flux balance analysis is a widely accepted constraint-based optimization tool for analyzing growth and metabolism, but it is generally used in a time-invariant manner with no provisions for sequestering different biomass components at different time periods. Here we present CycleSyn, a periodic model of Synechocystis sp. PCC 6803 metabolism that spans a 12-hr light/12-hr dark cycle by segmenting it into 12 Time Point Models (TPMs) with a uniform duration of two hours. The developed framework allows for the flow of metabolites across TPMs while inventorying metabolite levels and only allowing for the utilization of currently or previously produced compounds. The 12 TPMs allow for the incorporation of time-dependent constraints that capture the cyclic nature of cellular processes. Imposing bounds on reactions informed by temporally-segmented transcriptomic data enables simulation of phototrophic growth as a single linear programming (LP) problem. The solution provides the time varying reaction fluxes over a 24-hour cycle and the accumulation/consumption of metabolites. The diurnal rhythm of metabolic gene expression driven by the circadian clock and its metabolic consequences is explored. Predicted flux and metabolite pools are in line with published studies regarding the temporal organization of phototrophic growth in Synechocystis PCC 6803 paving the way for constructing time-resolved genome-scale models (GSMs) for organisms with a circadian clock. In addition, the metabolic reorganization that would be required to enable Synechocystis PCC 6803 to temporally separate photosynthesis from oxygen-sensitive nitrogen fixation is also explored using the developed model formalism. Flux balance analysis (FBA) has become a popular tool to analyze the metabolic function of organisms [1]. FBA assumes the cell is operating at a pseudo steady-state, wherein for each internal metabolite the sum of production fluxes must equal the sum of consumption fluxes. The steady-state assumption hinges upon the requirement that the time constants characterizing metabolic reactions are very rapid compared to the time constant of cell growth [2]. This time flux invariance places tight constraints on the feasible solution space and underpins the explanatory and predictive success of FBA [3–5]. However, for many organisms temporal and periodic variations in metabolism are part of their lifestyle [6]. This is the case for phototrophic organisms whose metabolism is tailored around light availability over a 24-hour cycle. Two distinct phases can be identified here: a light phase that centers around synthesis of metabolic precursors and storage compounds, and a dark phase that consumes those storage compounds to ensure survival in the absence of an energy source [7]. The transition between these two phases is driven by the circadian clock that choreographs the temporal expression of thousands of genes [6]. Highly varying gene expression levels over the 24hr cycle implies that the corresponding metabolic fluxes would also vary significantly and the biomass precursor production be dynamically shaped as the cumulative contribution by metabolism over 24 hours. FBA describes metabolic fluxes as the average over the 24hr period thus missing the opportunity to describe the (i) temporally varying nature of metabolism, (ii) time dependent inventory and remobilization of metabolites, and (iii) the time when different components of biomass are produced. This implies that FBA needs to be augmented so that it can accommodate temporally varying gene transcription information while still permitting the use of the pseudo steady-state assumption, by exploiting the difference in time-scales between metabolic reactions and cell growth. In their natural habitat, cyanobacteria are subject to a diurnal cycle of light and dark, leading to significant shifts and reorganization within their metabolic network. Although several studies, both experimental and computational [8–10], have helped to illustrate this cyclic cyanobacterial lifestyle, metabolic studies have primarily focused on conditions of constant illumination or heterotrophic growth on externally-supplied carbon sources. Kinetic models of cyanobacterial metabolism can capture the temporal biochemical interactions in the system, but are only available for select subsystems, such as the cyanobacterial circadian clock [11,12], photosystem II [13], and the Calvin-Benson cycle [14,15]. These temporal transitions cannot be described using conventional FBA, and these limitations have been recognized before. Knoop et al. [16] augmented FBA by introducing a time varying biomass composition tailored around light availability. For instance, the ratio for pigments in the biomass reaction was increased two hours before sunrise and storage compound coefficients increased after noon. A set amount of glycogen was supplied to the model for fueling dark respiration instead of transferring the storage compounds generated during light to dark [16]. Optimal temporal allocation of resources has also been employed as a tool to model diurnal lifestyles using an approach called conditional FBA [17,18]. Conditional FBA limits the flux through a reaction by accounting for the abundance of enzymes (or enzyme complexes) and their catalytic turnover numbers [17,18]. Nonlinear constraints are used to maintain periodicity which makes the size of the model grow as the square of the number of time steps being simulated [17,18]. The LP problems solved at each iteration may also become ill-conditioned due to the orders of magnitude differences in fluxes. Finally, required inputs such as enzyme turnovers are often hard to determine accurately. CycleSyn alleviates these challenges by recasting diurnal growth as a single linear optimization framework, with solve times of the same order as that of a standard FBA. A 12h light/12h dark cycle is discretized into twelve intervals, each of which abides by the pseudo-steady state hypothesis, to provide twelve snapshots of metabolism (see Table 1 for a comparison of CycleSyn with other published models of dynamic metabolism). By connecting these snapshots by metabolite transfer reactions, CycleSyn also provides insights into metabolite accumulation and consumption which are in line with published literature [19,20]. The only model input apart from model stoichiometry and biomass composition is transcriptomic data collected at 2-hour intervals, which is used to throttle back the upper bounds of corresponding reactions. Apart from modeling phototrophic metabolism, CycleSyn can be applied to functions typically served by conventional FBA such as testing in silico knockout mutants or identifying essential reactions/pathways under a varying light regime. In the absence of omics data from mutant strains, algorithms such as RELATCH [21] can be used in conjunction with CycleSyn to predict the effect of gene knockouts. The primary assumption employed there is that perturbed strains minimize relative metabolic changes and increase the capacity of previously active and inactive pathways in order to adapt to perturbations. By employing flux and gene expression data from wild-type strains, Kim et al. were able to successfully predict flux distributions in genetically and/or environmentally perturbed E. coli, S. cerevisiae, and B. subtilis strains. The current text demonstrates the ability of CycleSyn to guide the redesign of temporally-varying metabolism by identifying the metabolic shifts required to incorporate diazotrophy in a phototroph such as Synechocystis sp. PCC 6803 (hereafter referred to as Synechocystis). In this paper, we seek to capture the temporal changes in phototrophic metabolism over a diurnal cycle by modelling a 24-hour day as twelve individual Time-Point Models (TPMs), with each TPM spanning a two-hour period during 12 hours of light and 12 hours of dark. The pseudo-steady state assumption of standard FBA is imposed at every TPM. Metabolite balances are imposed at every TPM though accumulation and/or net consumption of metabolites is allowed. Any metabolite surplus in the cytosol or carboxysome is transferred to the next TPM. Metabolite levels are not allowed to drop below zero implying that all metabolite consumption within a TPM must not exceed the surplus provided by the previous TPM and the amount produced in the current TPM. Reaction flux upper bounds are set in proportion to the temporally varying transcriptomic data. The cascade of TPMs satisfies periodicity constraints by matching the output from the 12th TPM with the input to the 1st one. Comparisons with experimental observations for Synechocystis cultured under a 12h/12h light-dark cycle are used to ascertain biological fidelity. We find that CycleSyn correctly predicted the accumulation of metabolites such as glycogen, the primary storage compound in Synechocystis, and was able to replicate the temporal variations in metabolic pathways as seen in a diurnally cultured Synechocystis. We also found that upon constricting reaction fluxes using temporally segmented transcriptomic data, the primary bottlenecks in wild-type Synechocystis biomass production centered around pyruvate and 2-oxoglutarate metabolisms. Upregulating their production and/or diverting pyruvate flux selectively into the TCA cycle would lead to increased growth, as has been experimentally observed in Synechococcus elongatus PCC 7942 [27]. Subsequently, we used the 24hr model to address the metabolic flux rewiring needed to enable nitrogen fixation in a temporally segregated manner from photosynthesis in Synechocystis. We found that the added energy needed to fuel nitrogen fixation needs to be supplied by an enhanced TCA cycle turnover together with an upregulation of photosynthesis and glycolysis. The genes that need to be upregulated with respect to a non-diazotrophic wild-type Synechocystis are associated with pathways of energy metabolism, so as to meet the higher energy requirements posed by nitrogen fixation and amino-acid production. The 24-hour model was created starting from the published Synechocystis genome-scale model (GSM) iSyn731 [28] as a reaction source, which was updated to include the latest Synechocystis genome annotation from CyanoBase (http: //genome. annotation. jp/cyanobase/Synechocystis). Twelve separate GSMs (each called a Time Point Model or TPM) (Fig 1), each spanning a two-hour period starting from the first light time point (L0-L2) to the last dark time point (D10- D12) were linked. Initially, all TPMs are the same except that TPMs 1 through 6 are allowed to take up light as photons and carbon as carbon dioxide whereas TPMs 7 through 12 are not. The maximum CO2 uptake rate was set to 1. 1 mmol CO2 g-1 dry weight hr-1 for every light TPM, as cyanobacteria are known to not uptake carbon during dark [29–32] and no fixation occurs in the dark due to the lack of photons. This CO2 uptake flux corresponds 0 to 13 mg-1 dry weight hr-1 of carbon [33,34]. A basal ATP maintenance demand was also set for every TPM at 10 mmol g-1 dry weight h-1 [28]. The TPMs are connected by the unidirectional forward transfer of metabolites present in the cytosol and carboxysomes, thus only allowing for the consumption of a metabolite in a specific TPM if the metabolite was previously produced or is produced during the current TPM. Any metabolite surplus in the cytosol and carboxysome except photons and protons are transferred to the next TPM (see Materials and Methods). The cyclic topology of the TPMs implies that metabolic flux can go around a closed loop forming a thermodynamically infeasible cycle [35]. To remedy this, the sum of flux through all transfer reactions (between TPMs) is minimized using modified parsimonious flux balance analysis (pFBA) [36] after constraining the biomass production flux to its theoretical maximum. This is implemented in CycleSyn as an additional model constraint. It should be noted that even though the transfer of energy metabolites such as ATP, NAD (P), and NAD (P) H is allowed, CycleSyn results retain their qualitative trends when their transfer fluxes were set to zero. This is because transferring a single storage molecule such as glycogen to satisfy energy demands during the dark phase is preferred by CycleSyn as it is more in line with the model constraint of minimizing the sum of all transfer fluxes. Photosynthesis in the model is coupled to chlorophyll availability by only allowing flux through photosynthesis reactions in a TPM if chlorophyll is present in that TPM, as coupling chlorophyll production to photosynthetic flux places additional demands on chlorophyll synthesis outside of serving as a biomass precursor. This additional demand is corroborated by matching predicted photosynthetic oxygen evolution flux to experimental values (1) (see Materials and Methods). A single biomass sink placed in the last dark TPM (i. e., TMP12) sequesters all biomass components in the experimentally measured ratios to model growth, although CycleSyn results were similar when the biomass drain was placed in TPM6. TPM6 was chosen as a test case as it is known that very little biomass is produced in the dark [37]. It is important to note that the production of different biomass components is apportioned in a non-uniform manner over the twelve TPMs and only in the last TPM are their fluxes combined to form biomass (Fig 1). The periodicity in metabolism is captured by using transcriptomic data collected over two-hour intervals to constraint reaction fluxes [38]. Specifically, the upper flux bound of a reaction was scaled as a function of its associated gene expression value normalized by the maximum expression over all TPMs, thereby constricting the maximum allowable flux through it. Each reaction’s unscaled flux bounds are determined using only stoichiometric and thermodynamic constraints in order to determine the largest feasible flux range (see Materials and Methods). This approach is often referred to as the valve approach of regulation [39]. The predicted biomass production flux before adding transcriptomic constraints was 0. 03 hr-1 which corresponds to an average doubling time of 22. 8 hours [40]. This closely aligns with the experimentally determined doubling time of wild-type Synechocystis under phototrophic conditions, which is approximately 24 hours (~0. 0288 hr-1) [28]. Following the scaling of reaction fluxes using their corresponding transcriptomic ratios, the maximum biomass production flux was reduced by a tenth of its original unconstrained value. This corresponds to a doubling time of ~25 hours. Doubling times in the range of 20–40 hours have been seen for diurnally cultured Synechocystis [41]. CycleSyn uses reaction bounds informed from transcriptomic data to distinguish among the individual light and dark TPMs by relatively throttling reaction upper bounds based on gene expression. Implicit to this is the assumption that RNA levels track protein levels. This correlation between gene expression and protein levels has been shown both experimentally and computationally before [42–45]. Notably Zelezniak et al. [46] found that the correlations between gene expression and metabolite concentrations increases when considered against a background of a metabolic network. We assessed the effect of introducing normally-distributed white noise (within one standard deviation of the mean of the gene expression normalized over time) in the connection between gene expression and proteins. We found that CycleSyn predictions were within 19. 28% of their unperturbed flux values, when averaged over all metabolites and all time points. Different light-sensing proteins help mediate external light cues to coordinate inner metabolic processes. Studies in cyanobacteria such as Cyanothece ATCC 51142 (hereafter referred to as Cyanothece) have shown that the abundance of many proteins change over the diurnal period [47]. 40. 3% of these proteins are associated with central metabolism and energy pathways and 18. 5% were associated with photosynthesis and respiration [47]. A majority of Synechocystis genes also show cycling, with the peak expression of cycling genes being during the transition from day to night, regulating energy supply and carbon metabolism [48]. Here, we examine if the diurnal nature of a cellular process was maintained from the gene to the metabolic level by incorporating gene expression data using the E-Flux method [39] (see ‘Materials and Methods’ section). Using a modified parsimonious flux balance analysis (pFBA) [36] to minimize the total sum of all fluxes through the metabolite transfers between each TPM, the transfer flux between TPMs was predicted. The transfer flux for a metabolite from one TPM to the next can be interpreted as the accumulation/consumption of that metabolite in that particular TPM. This allows us to compare the model-predicted metabolite accumulation to the experimentally measured metabolite levels in a diurnally cultured Synechocystis. As the LP has multiple alternative optima, flux variability analysis [49] was used to determine the maximum and minimum possible transfer flux between TPMs and used to construct Fig 2. This involves minimizing and maximizing the flux through the metabolite transfer reactions while ensuring that the model continues to produce biomass at the maximum value possible and the sum of all transfer fluxes does not exceed the value obtained using the aforementioned modified pFBA. The maximum and minimum transfer flux was taken for every transferring metabolite and summed over to assess the distribution across categories (see supplementary S6 Table for a list of metabolites and their classifications). For metabolite classes such as amino acids, pigments, organic acids, and energy metabolites, there is very little variability between the maximum and minimum transfer flux across TPMs. This is due to the limitations placed on the maximum allowable flux through a reaction using transcriptomics data and the modified pFBA model constraint that further minimizes the sum of all transfer reactions, thus reducing variability. Metabolites are only allowed to accumulate so as to satisfy the demands placed on the system, such as biomass production and ATP maintenance. The amino acid flux profile, governed mainly by glucogenic amino acids such as proline and alanine, is found to be at its maximum during early light with a sharp decline at the transition between light and dark, i. e. between TPM6 and TPM7. This points towards the role of proline as a carbon and nitrogen reservoir, as is known to occur in Cyanobacteria [50,51]. CycleSyn predicts that proline flux feeds into glutamate biosynthesis during the light TPMs by the action of glutamate dehydrogenase. This is corroborated in literature, where glutamate concentrations are known to increase during light in a diurnally cultured Synechocystis [41] and the NADP-specific glutamate dehydrogenase functions only during light in the phototroph C. sorokiniana [52]. The decrease in amino acid transfer flux during the transition from light to dark can be explained by the concurrent increase in accumulation of energy metabolites such as ATP, GTP and CTP. Glucogenic amino acids are degraded towards TCA cycle intermediates such as oxaloactetate, thereby producing energy equivalents in the form of ATP. Glucogenic amino acids levels are known to decrease right after the transition from light to dark incubation in wild-type Synechocystis [20], alongside a substantial upregulation in genes involved in ATP synthesis [53]. This transition from light to dark is also accompanied by an increase in flux through the oxidative part of the pentose phosphate pathway (OPPP). OPPP is the major pathway of glucose catabolism in heterotrophic and mixotrophic cultures of Synechocystis [54] and is known to be used in conjunction with the Calvin cycle to regulate carbon fixation in autotrophic conditions [54]. The initiation of photosynthesis after the transition from light to dark uses energy compounds such as NADPH, ATP, and Calvin cycle intermediates. As these intermediates are shared with glycolysis, it is expected that respiration during dark time periods be closely linked to the initiation of photosynthesis in diurnally-growing Synechocystis [56]. Glycogen is the primary respiratory substrate in Synechocystis and its degradation is initiated by glycogen phosphorylase transferring orthophosphate to the non-reducing end of the glucose residue in glycogen and releasing glucose-1-phosphate, which feeds into glycolysis. Experiments with Synechocystsis mutants deficient in glycogen phosphorylase (ΔGlgP) found that the amount of dark respiration was 25% lower than that in the wild-type, following which the photosynthetic oxygen evolution rate reached its steady-state value at a later time [56], delineating a temporal dependence between glycolysis during the dark and photosynthesis during light. Our simulations also show accumulation of circulating Calvin cycle intermediates during the dark TPMs. Metabolites such as 3-phosphoglycerate, which is used to regenerate D-ribulose-1,5-bisphosphate (RuBP) during photosynthesis [57], and other glycolytic metabolites such as 2-phosphoglycerate and fructose-6-phosphate exhibit this phenomena. These metabolites are fed into glycolysis during the dark and enter the Calvin cycle during the transition from dark to light, suggesting that glycolytic intermediates produced during respiration in the dark are used for regenerating RuBP via the Calvin cycle during the induction of photosynthesis. Interestingly, the total accumulation of RuBP was higher in light than in the dark, implying preferential production and degradation of metabolites guided by the organism’s circadian clock. This is in alignment with cyanobacteria upregulating the oxidative pentose phosphate pathway in the absence of light [58]. Metabolite classes such as fatty acids, porphyrins, nucleotides, and proteins were selectively produced in the light as opposed to the dark TPMs, in agreement with literature. Fatty acid biosynthesis is known to increase with increasing light intensity in Synechocystis [59] and the responsible enzymes involved show increased synthesis during the light in Cyanothece as well [60]. Furthermore, photo-oxidative stress during photosynthesis gives rise to reactive oxygen species and initiates redox signaling. Ansong et al. [61] showed that several proteins involved in fatty acid biosynthesis are redox controlled in Synechococcus elongatus 7002, including acetyl CoA-carboxylase, which catalyzes the first step of fatty acid biosynthesis. Nucleotide and protein metabolism enzymes were shown to have the highest representation among all redox sensitive proteins, both of which show higher metabolite accumulation in light as opposed to dark in CycleSyn (Fig 2). Increased protein accumulation during light is also supported by an upregulation in the corresponding genes involved in protein synthesis in Synechocystis [38]. The increase in porphyrins such as heme during the light TPMs is the consequence of the increase in pigment production. Heme and chlorophyll are both tetrapyrrole pigments and hence share a common biosynthetic pathway [62]. Both chlorophyll and heme production is known to be upregulated during light in cyanobacteria [38,60], due to their central role in photosynthesis [63]. Interestingly, terpenes such as lycopene and gamma-carotene are synthesized in the latter half of the day in CycleSyn, rising just before the transition to dark and maintaining these levels throughout the dark TPMs. We sought to investigate the source of this production using Metabolite-metabolite correlation analysis (MMCA) [64–67] (see Fig 3), which assesses metabolite concentration interdependencies using similarity metrics. These dependencies have been used before to explore the temporal organization of metabolic networks [68,69]. The analysis herein calculates pair-wise correlation coefficients between time-resolved transfer flux profiles for all metabolites using the non-parametric Spearman Test, employing a two-tailed test for hypothesis testing with a p-value cut-off of 0. 05. The transfer flux for every metabolite was normalized with respect to the maximum value recorded across all TPMs and used as a proxy for metabolite concentrations. MMCA is usually employed on experimental datasets of metabolite concentrations, but CycleSyn’s ability to predict metabolite accumulation levels under a FBA paradigm enables us to use MMCA to study possible correlations between metabolite transfer fluxes. MMCA showed that metabolites of the pentose phosphate pathway such as Ribulose-1,5-bisphosphate (RuBP) and dihydroxyacetone phosphate (DHAP) are negatively correlated with terpenes such as gamma-carotene, beta-carotene, and lycopene, the three terpenes showing maximum production during late light (Fig 3), thus indicating that a rise in the levels of terpenes is associated with a fall in the levels of RuBP and DHAP. A similar phenomenon has been observed earlier by Ershov et al. [70] for a phototrophically growing Synechocystis, where terpenoid biosynthesis was stimulated by the addition of DHAP and other compounds of the pentose phosphate pathway, such as glyceraldehyde-3-phosphate (G3P) and D-ribulose 5-phosphate. Isoprenoid synthesis in Synechocystis occurs via the 2-C-methyl-D-erythritol pathway (MEP pathway), which starts with the condensation of glyceraldehyde 3- phosphate (GA3P) and pyruvate [71]. Thus, the substrates for terpenoid production are obtained from the metabolite products of photosynthesis such as DHAP and G3P. This also explains the temporal order of terpenoid production, wherein products of photosynthesis need to accumulate in order for the MEP pathway to be active, thus leading to terpene production in the late light, as replicated in CycleSyn. Furthermore, genes associated with the MEP pathway were seen to be upregulated in the dark in a diurnally cultured Synechocystis, such as those corresponding to phytoene dehydrogenase, phytofluene dehydrogenase, and the reaction CTP: 2-C-Methyl-D-erythritol 4-phosphate cytidylyltransferase [38], which catalyzes the conversion of MEP into 4- (cytidine 5’-diphospho) -2-C-methyl-D-erythritol and constitutes the second step of the MEP pathway. The metabolite class made up of RNA was found to be largely insensitive to the diurnal changes in metabolism, transfer fluxes ranging between 4. 345 and 4. 328 mmol gDW-1 hr-1. The time invariant nature of RNA production is consistent with a study that determined the total amount of tRNAs is relatively constant over a diurnal cycle in Synechocystsis, with the major RNA variability originating from long transcripts such as 16S rRNA and 23S rRNA [72] which are not captured in the present metabolic reconstruction. Carbon fixation in Synechocystis during light exceeds needs (luxury uptake [73,74]) so as to catabolize those reserves in the dark to support growth and cellular maintenance. Glycogen is employed as such a reserve in Synechocystis [75,76], providing maintenance energy for cellular functions during dark periods [77]. As seen in Fig 4 and S1 Table, glycogen accumulates during the day and is gradually consumed during the night in CycleSyn. The highest glycogen transfer flux was recorded at the transition between light and dark in our simulations. Fig 4 contrasts the experimentally observed glycogen concentrations with the simulated glycogen accumulation levels. This comparison enables us to approximate the amount of a metabolite shuttled across TPMs after all its production/consumption reactions have taken place, so as to examine its overall temporal dynamics and contrast it with experimental values. Model-predicted glycogen dynamics is in accordance with experimental data, with the total glycogen content increasing gradually during light and reaching its peak level just before the onset of dark. The dark TPMs see a progressive decrease in glycogen as it is utilized as a respiratory substrate [77]. In particular, CycleSyn glycogen accumulation during light matches with the experimental levels seen by Angermayr et al. [41], but unlike Angermayr et al. we do not see a biphasic decline during late dark, which is also consistent with earlier studies [20,38,78]. Angermayr et al. [41] attribute the rapid decline in glycogen during the last two hours of dark to increased acetate accumulation and an upregulation of genes encoding the bidirectional NiFe-hydrogenase that is thought to help maintain the redox balance by reducing H+ [79]. CycleSyn did not predict a higher flux through NiFe-hydrogenase during the dark. In order to further ascertain the veracity of the model-predicted glycogen levels, we also compared the glycogen accumulation in the dark TPMs to data from Hanai et al. [20] (Fig 4). In this study Synechocystis was cultured under a 12hour light/12hour dark cycle and the glycogen concentration (as nmol per gm fresh weight (FW) ) was measured at 0,2, 6, and 12 hours after the transition to dark (Fig 4). As seen in Fig 4, CycleSyn predicted glycogen dynamics during the dark matches that seen by Hanai et al. [20]. Although CycleSyn predicts glycogen accumulation during light and degradation during the dark, the minimum possible transfer flux for glycogen is zero, indicating that other metabolites can serve as additional storage reserves. Isocitrate is found to be such a possible storage metabolite that is accumulated during light and degraded in the dark TPMs. Its catabolism occurs via isocitrate dehydrogenase, encoded by the icd (slr1289) gene which has been found to be upregulated in the dark as compared to light in a diurnally cultured Synechocystis by Saha et al. [38]. Experiments with Synechocystis sp. PCC 6803 impaired in glycogen synthesis have displayed overflow of pyruvate and 2-oxoglutarate [80,81], suggesting that carbon excess is directed preferentially into these compounds in the absence of glycogen. Isocitrate dehydrogenase catabolizes isocitrate to produce 2-oxoglutarate, whose central role in Synechocystis metabolism is discussed below and elucidated through metabolic control analysis. The biomass equation approximates the composition of dry biomass and is used to drain biomass precursors in their physiologically relevant ratios. Fig 5 describes the sum of the maximum and minimum transfer fluxes for classes of biomass precursors over a 24-hour diurnal cycle (see S1 Table for the list of transfer fluxes of all biomass precursors). As seen in Fig 5 and S1 Table, biomass precursors such as carbohydrates and nucleotides are primarily produced during the day and sequestered in the night during the last TPM. Chlorophyll is synthesized during light as is known to occur in cyanobacteria [41], with these levels remaining constant during the dark TPMs. By constraining reaction flux using transcriptomic data, we identified 110 reactions (see S2 Table) with active bounds, i. e. reactions whose flux constraints limit metabolism when the biomass objective function is maximized. This also resulted in a decrease in the biomass flux, which dropped by 10% (compared to the unconstrained flux case) corresponding to a doubling time of ~25 hours. As transcriptomic constraints induced a decrease in the biomass production flux, alleviating some or all of these bounds might allow for an increase in the maximal biomass produced. The 110 reactions with active bounds tend to maintain active bounds in multiple TPMs. After adjusting for this reoccurence, 33 unique reactions were identified—one exclusively in dark TPMs and the rest during light TPMs. The reactions with active upper bounds belong mainly to central carbon and amino-acid metabolism, alongside peripheral metabolic pathways such as purine, pyrimidine, aminosugars, and lipid metabolism, and can be broadly linked to pyruvate and 2-oxoglutarate metabolism. Reactions involved in central carbon metabolism such as glycolysis and the pentose phosphate pathway also have active constraints, such as the conversion of 3-phosphoglycerate to 1,3-bisphospho-D-glycerate and the reaction between ribose-5-phosphate and D-xylulose5-phosphate to produce glyceraldehyde-3-phosphate and sedoheptulose-7-phosphate. Furthermore, reactions belonging to glucogenic amino acid metabolism are also found to have active reaction bounds. Glucogenic amino acids such as lysine and aspartate yield through catabolism pyruvate or TCA cycle metabolites. As the TCA cycle is the primary source of ATP in cyanobacteria, upregulating these reactions during early light would allow for a greater TCA cycle turnover, leading to more biomass production and hence enhanced growth. The need to increase 2-oxoglutarate production and thus TCA cycle turnover is further evidenced by the constriction of reactions such as L-Phenylalanine: 2-oxoglutarate aminotransferase, L-Aspartate: 2-oxoglutarate aminotransferase, L-Valine: 2-oxoglutarate aminotransferase, and L-Leucine: 2-oxoglutarate aminotransferase in the direction of 2-oxoglutarate production. Enhanced pyruvate production has led to greater biomass production in cyanobacteria in an earlier study [27]. Modifying glycolytic pathways and the Calvin Benson cycle in Synechococcus elongatus PCC 7942 to redirect flux towards carbon fixation increased the intracellular pool of pyruvate, which led to about a three-fold increase in growth under both light and dark conditions [27]. In order to further investigate the central roles played by pyruvate and 2-oxoglutarate, we used metabolic control analysis [82] to identify reactions that most affect the biomass production upon a perturbation in their corresponding enzyme levels. A 1% enzyme perturbation was considered and the flux control coefficient (FCC) calculated for every reaction using transcript levels as proxies for the enzyme levels [83] (see Materials and Methods). FCCs provide a quantitative measure of the degree of control a particular enzyme exerts on the reaction flux of interest. Interestingly, of all the reactions considered, only two affected the final biomass production flux and only during light TPMs. These included reactions L-Tyrosine: 2-oxoglutarate aminotransferase and L-Phenylalanine: 2-oxoglutarate aminotransferase with FCCs of 0. 016 and 0. 04, respectively. Both these reactions are controlled by the same set of isozymes in Synechocystis which are not shared by any other reaction and both bidirectional reactions operate selectively in the direction of 2-oxoglutarate synthesis, alluding to the importance of 2-oxoglutarate in Synechocystis growth. The intracellular concentration of 2-oxoglutarate in Synechocystis has been implicated in the regulation of the coordination of carbon and nitrogen metabolism [84]. As Synechocystis lacks the traditional 2-oxoglutarate dehydrogenase complex, 2-oxoglutarate acts as the final carbon skeleton for nitrogen. It is used to sense changes in the cell’s nitrogen status [85] and provides the carbon backbones needed for synthesizing amino acids such as glutamate, glutamine, proline, and arginine biosynthesis via the GS-GOGAT cycle [86]. Nitrogen Fixation was introduced to the model by including the relevant reactions from iCyt773, the GSM model for Cyanothece [28], and constraints to ensure that anoxic conditions are maintained during nitrogen fixation (see Materials and Methods), as the nitrogen-fixing enzyme nitrogenase is irreversibly inhibited by oxygen. Transcriptomic data from wild-type Synechocystis [38] was used to restrict reaction flux bounds, so as to identify the reactions that need to be regulated differently in order to fix nitrogen while maintaining growth. S3 Table lists the 672 reactions with flux ranges that change compared to the earlier-described diurnally growing wild-type Synechocystis (i. e. the feasible flux range associated with these reactions do not overlap with the flux range associated with the wild-type strain). 236 reactions (107 unique reactions after adjusting for TPM multiple participation) were upregulated and 436 reactions (225 unique) were downregulated upon the introduction of nitrogen fixation. A total of 166 reactions (128 unique) were found to be essential under diazotrophic conditions, i. e. had strictly positive or strictly negative flux profiles, out of which 44 reactions were non-essential under wild-type conditions. These reactions are indicators of the metabolic alterations required as a result of the introduction of diazotrophy in Synechocystis (see Fig 6 and S3 Table). Reactions with a perturbed flux profile mainly belong to pathways of carbon and amino-acid metabolism, and target the two important modifications required for nitrogenase to function–increased ATP availability and an anaerobic environment. Upregulation of reactions such as L-Aspartic acid: oxygen oxidoreductase help maintain the latter, while the former is addressed by reactions belonging to glycolysis, TCA cycle, and photosynthesis in the light TPMs, i. e. TPMs 1 to 6, glycogen synthesis, and oxidative pentose phosphate pathway in the dark TPMs, i. e. TPMs 7 to 12 (Fig 7 and S3 Table). As nitrogen fixation is an energy-intensive process, requiring 16 ATP molecules and eight reducing equivalents for every molecule of dinitrogen fixed, this energy is being provided by the coordinated actions of these pathways, while the required reducing equivalents are being supplied by the upregulation of Ferredoxin: NADP+ oxidoreductase reaction, which converts NADP to NADPH while oxidizing ferredoxin. A similar phenomenon is seen in a diurnally cultured Cyanothece, where transcriptomic analysis shows simultaneous upregulation of entire pathways involved in respiration and energy metabolism, such as pentose phosphate pathway, TCA cycle, glycolysis, and amino-acid metabolism in the dark [87]. Cyanothece emerges as a natural comparison for the hypothesized diazotrophic Synechocystis as in order to accommodate both nitrogen fixation and photosynthesis, Cyanothece temporally separates the two incompatible processes [88]. A number of reactions with highly changed flux ranges are similar to the temporal distribution of flux seen in the nitrogen-fixing Cyanothece. For instance, there is an increase in flux in the reactions responsible for glycogen degradation during late dark, which is fed into glycolysis via glucose-1-phosphate, so as to fuel the higher energy demands associated with nitrogen fixation. Similar to the diazotrophic Cyanothece, glycogen degradation in CycleSyn proceeds via glycolysis, the oxidative pentose phosphate pathway, and the TCA cycle so as to provide the cell with ATP, cellular precursors, and pyrimidine nucleotides. Increased respiration of carbohydrate reserves in the dark produces NADPH and succinate, which transfers electrons via NADPH dehydrogenase and succinate dehydrogenase into the plastoquinone pool [90], towards the terminal electron acceptor. This electron transport due to respiration sets up a proton gradient and drives ATP production. The increased flux towards succinate is evinced by the upregulation of (S) -Malate hydrolyase in the dark TPMs which reversibly converts malate into fumarate [47]. A similar upregulation is seen in Cyanothece BG 043511 as well, where an increase in nitrogen fixation at night coincided with a rise in respiratory electron transport [91]. Furthermore, as fixed nitrogen is incorporated via arginine and aspartate metabolic pathways, a number of reactions belonging to these pathways also show upregulation in the dark in a diazotrophic Synechocystis as opposed to the wild-type. Modelling phototrophic growth using constraint-based optimization techniques necessitates modeling contributions beyond conventional flux balance analysis. Since cyanobacteria show strong diurnal rhythms in its lifestyle, translating that phenotype into a metabolic model requires new approaches that enable us to incorporate and replicate those temporal metabolic reorganizations. Diurnal oscillations in Cyanobacteria have been the focus of many studies [6,17,53] but those have mainly been concentrated on the associated transcriptomic changes or a subset of its entire metabolism. In this work, we bridge that gap by developing a diurnal model of Synechocystis metabolism. We accommodated the cyanobacterial circadian clock and its influence on the underlying metabolic machinery by employing temporally-resolved transcriptomic data. The developed formalism was able to replicate the changes in metabolism observed in Synechocystis over a diel light-dark cycle. It should be noted here that CycleSyn does not assume that metabolite concentrations, enzyme activities, or reaction rates are governed solely by mRNA expression levels. It is well known that the true flux through a reaction depends on the enzyme kinetics and expression, alongside metabolite concentrations (Michaelis-Menten kinetics). The biological rationale behind CycleSyn is that expression data provides measurements of the level of mRNA available for each gene. If there was a limited accumulation of an enzyme in a particular TPM with respect to the others, the (relative) level of mRNA can be used as an approximate upper bound for the maximum protein available. This can then be used to constrict the maximum permissible flux through a reaction, effectively reshaping the metabolic flux cone. This enables a systematic extension of flux balance analysis by making use of temporal changes in expression levels to predict the metabolic capacity of Synechocystis over a 24-hour period. The correspondence between transcript levels, reaction fluxes and metabolite levels has been elucidated before [92] where accuracy in predicting the direction of change (increase/decrease) in metabolite levels increased by 90% when constraints derived from transcriptomic data were included in the metabolic model of a maize leaf. The 24hr model provides a time-course for all reaction fluxes and metabolite levels. The model predictions are aligned well with several known phenomena in a diurnally-controlled cyanobacterial phototrophic metabolism. We predicted that glycogen was accumulated during light and degraded steadily during dark, as is seen in Synechocystis [38,41]. Different pathways were upregulated during the light and dark phases, highlighting the variations in metabolism occurring due to light availability. Glycolysis intermediates produced during respiration in the dark were being used for regenerating RuBP via the Calvin cycle during the induction of photosynthesis. Levels of amino acids produced from glycolytic precursors such as pyruvate and alpha-ketoglutarate decreased at the transition from light to dark incubation in wild-type Synechocystis alongside a substantial upregulation in genes involved in ATP synthesis, as is seen experimentally [20], [53]. CycleSyn also predicted pyruvate metabolism as a bottleneck in biomass synthesis. Redirecting flux towards pyruvate synthesis can increase carbon fixation and hence biomass formation, as is seen in Synechococcus elongatus PCC 7942 [27]. We also treated Synechocystis as an example cyanobacterium to predict the various metabolic pathways that would need to be regulated in a photosynthetic organism so as to incorporate the two inherently incompatible processes of photosynthesis and nitrogen fixation. The introduction of nitrogen fixation drew parallels from the non-heterocystous cyanobacteria Cyanothece ATCC 51142, which temporally separates the two antagonistic processes. In doing so it fixes glycogen during the day and uses it as a respiratory product in the dark, thus also achieving the anoxic conditions required for nitrogenase activity. This necessitates a reorganization of the cellular metabolic processes, with dominant flux-carrying pathways differing during the light and dark periods. The model predicted changes in pathways of carbon fixation and amino acid synthesis upon introduction of diazotrophy in Synechocystis. The dark phase in Cyanothece is known to have a high protein turnover, with upregulation of amino-acid biosynthesis pathways due to the increased nitrogen sequestration. Pathways such as arginine and aspartate metabolism were consequently upregulated in the dark, due to the need to sequester the fixed nitrogen. CycleSyn predicted the high-energy demands associated with nitrogen fixation to be met by increased flux through TCA cycle and the pentose phosphate pathway, maintained by higher glycogen synthesis and remobilization. Furthermore, oxygen scavenging reactions such as L-Aspartic acid: oxygen oxidoreductase were upregulated across dark TPMs, due to their oxygen-scavenging role which is required to maintain the anaerobic conditions required for nitrogenase to function. The developed framework enables analyzing a time-variant GSM model while preserving the fundamental time-invariant assumption of conventional flux balance analysis. It improves upon existing techniques of diurnal simulations of metabolism while maintaining a linear programming problem resulting in low computational costs. The formulation can readily be customized to accommodate quantitative measurements of reaction fluxes over a 24-hour cycle. This will also constrain the feasible solution space and thereby improve the precision and accuracy of model predictions [93]. We expect this and similar methods to become instrumental in understanding, analyzing, and predicting temporal metabolic flux variations. We constructed CycleSyn using the iSyn731 genome-scale metabolic (GSM) model for Synechocystis sp. 6803 [28] as a scaffold. iSyn731 was updated to reflect the latest annotations made to the Synechocystis genome as present in CyanoBase (see supplementary S5 Table). Additions include the Entner-Doudoroff pathway, the phosphoketolase pathway, and the light-independent serine biosynthesis pathway [94–98], among others. Metabolite and reaction IDs were borrowed from ModelSEED [99] wherever possible. From 1,156 reactions and 1,003 metabolites, the model increased to 1,165 reactions and 1,008 metabolites. Flux variability analysis was performed on this model to ensure that it is free of any thermodynamically infeasible loops which can carry unbounded flux. The 24-hour model consists of 12 individual Time Point Models (TPMs), each approximating reaction fluxes over a 2-hour period. The first TPM covers the first two hours of the light period (L0-L2), the second TPM covering the next two hours of light (L2-L4), with the pattern continuing until TPM12 which contains the last two hours in the dark period (D10-L0). Each time-point model was made by duplicating reactions (by appending ‘_tpmX’ to reaction name, where X is the TPM number ranging from 1 to 12) and metabolites (denoted by appending ‘[tpmX]’ to the metabolite name) in the base model. A single biomass reaction occurs in TPM12 to account for organism growth. All metabolites except photons and protons that are present in the cytosol and carboxysome are transferred unidirectionally from the n to the n+1 TPM using transfer reactions. A transfer reaction j for a metabolite i in a TPM k always operates in the forward direction from TPM k to TPM k+1 such that metabolitei, k→transferj, kmetabolitei, k+1 Photosynthesis is only allowed to occur in a TPM k if it contains the necessary chlorophyll. The chlorophyll balance on a TPM equates the difference between total chlorophyll synthesis and degradation fluxes to the difference between the chlorophyll transfer fluxes that exit and enter the TPM. This is mathematically represented as: ∑vchlorophyllsynthesis, k−∑vchlorophylldegradation, k=vpigmenttransfer, ktok+1−vPigmentTransfer, k−1tok Where vPigment Transfer, k to k+1 refers to the flux through the chlorophyll transfer reaction from TPM k to TPM k+1. The chlorophyll transfer flux from a TPM k to TPM k+1 represents the cumulative chlorophyll accumulation (i. e., from TPM 1 to TPM k). We approximate this as a linearly increasing function with respect to time for a single TPM. Hence, the average amount of chlorophyll made in a TPM k can now be calculated as <vchlorophyll, k>=12 (vPigmentTransfer, ktok+1−vpigmenttransfer, k−1tok) The constraint levied on the model that couples photosynthesis to chlorophyll availability is expressed as: vPhotosynthesis, k≤12 (vPigmentTransfer, ktok+1−vpigmenttransfer, k−1tok) MC (1) where MC is a constant large enough not to constrain flux through photosynthesis reactions and k the Time Point Model. vPigment Transfer, k to k+1 refers to the flux through the chlorophyll transfer reaction from TPM k to TPM k+1. This implies that amount of chlorophyll available to carry out photosynthesis is equal to the difference between the amount transferred in to time point k and the amount transported out to point k+1, divided by two. A value of 1000 for MC predicts photosynthetic oxygen evolution (estimated by the output flux through the oxygen exchange reaction during the light TPMs) ranging between 173 to 169 mmol O2 (gm chlorophyll) −1 which is in the same order as that of wild-type Synechocystis. A wide range of values have been reported experimentally ranging from 225 mmol O2 (gm chlorophyll) −1 (hr) −1 [100] to 380 mmol O2 (gm chlorophyll) −1 (hr) −1 [101], precluding the matching of a single value. The set of photosynthetic reactions included in this constraint are cytochrome b6/f complex, cytochrome c oxidase cytochrome oxidase bd, Mehler reaction, photosystem I (plastocyanin), photosystem I (ferrocytochrome), photosystem II, and succinate dehydrogenase (periplasm) (see S4 Table for model reaction identifiers and reaction descriptions for reactions belonging to this constrained set). The optimization formulation used to determine maximum biomass production flux is maximizevbiomass, TPM12 (2) subject to ∑j∈JSijkvjk=0, ∀i∈I, k∈K (3) vCO2uptake, k≤1. 1, k=1, …, 6 (4) vPhotonuptake, k≤60, k=1, …, 6 (5) vATPmaintenance, k≥10, ∀k∈K (6) vjkLB≤vjk≤vjkUB, ∀j∈J, k∈K (7) 0≤vj, k≤10,000, ∀j∈JTransfer, k∈K (8) vPhotosynthesis, k≤12 (vPigmentTransfer, ktok+1−vpigmenttransfer, k−1tok) MC, ∀k∈K (9) where Sijk is the stoichiometric coefficient for metabolite i in reaction j and TPM k, vjkLB and vjkUB are the upper and lower flux bounds for reaction j in TPM k. I, J, and K denote the sets of total metabolites, reactions, and Time Point Models (TPMs), respectively, and JTransfer the set of all transfer reactions. Constraints (4) and (5) refer to carbon (as CO2) and photons being supplied to only the light TPMs. The maximum amount of photons supplied to a TPM are such that it is not growth-limiting but at the same time not in excess so as to not trigger light-sensitive reactions. vCO2uptake, k and vPhonton uptake, k represent the carbon and photon uptake reactions, and vATP maintenance, k is the ATP maintenance requirement for a TPM k. Every transfer reaction j in TPM k is constrained to have a non-negative flux by Eq (8). As all the individual metabolites are transferred (only forward) throughout all TPMs, they may give rise to cycles that can carry unbounded flux. Hence, to prevent this cycling, the sum of transfer fluxes was set to a scalar f that was identified by solving a modified pFBA formulation. Minimizef=∑k∈K∑j∈JTransfervjk (10) subject to ∑j∈JSijkvjk=0, ∀i∈I, k∈K vCO2uptake, k≤1. 1, k=1, …, 6 vPhotonuptake, k≤60, k=1, …, 6 vATPmaintenance, k≥10, ∀k∈K vjkLB≤vjk≤vjkUB, ∀j∈J, k∈K 0≤vj, k≤10,000, ∀j∈JTransfer, k∈K vPhotosynthesis, k≤12 (vPigmentTransfer, ktok+1−vpigmenttransfer, k−1tok) MC, ∀k∈K vBiomass, TPM12=vBiomass, TPM12max (11) where JTransfer is the set of all transfer reactions and vBiomass, TPM12max the maximum biomass production flux as determined by (2). In photosynthetic organisms such as Synechocystis, a proton gradient is generated during photosynthesis across the thylakoid membrane that drives ATP formation. Transferring this gradient across time points would result in an untenable way for storing energy outside of storage compounds. To this end, protons and photons were not transferred across TPMs. The following optimization model formulation is used to carry out flux variability analysis (FVA): Maximize/Minimizevjk (12) subject to ∑j∈JSijkvjk=0, ∀i∈I, k∈K vCO2uptake, k≤1. 1, k=1,.., 6 vPhotonuptake, k≤60, k=1,.., 6 vATPmaintenance, k≥10, ∀k∈K vjkLB≤vjk≤vjkUB, ∀j∈J, k∈K vPhotosynthesis, k≤12 (vPigmentTransfer, ktok+1−vpigmenttransfer, k−1tok) MC, ∀k∈K 0≤vj, k≤10,000, ∀j∈JTransfer, k∈K vBiomass, TPM12=vBiomass, TPM12max ∑k∈K∑j∈JTransfervjk≤f (13) CPLEX solver (version 12. 1, IBM ILOG) was used in the GAMS (version 23. 3. 3, GAMS Development Corporation) environment for solving all optimization models. All computations were carried out on dual 10-core and 12-core Intel Xeon E5-2680 and Intel Xeon E7-4830 quad 10-core processors that are the part of the ACI cluster of High Performance Computing Group of The Pennsylvania State University. Numerical scaling issues were not observed when solving CycleSyn. | Title: A diurnal flux balance model of Synechocystis sp. PCC 6803 metabolism Summary: Phototrophic organisms such as cyanobacteria harvest the sun's energy to convert atmospheric CO2 into organic carbon, due to which their metabolism is heavily influenced by light availability. The strongly diurnal nature of their metabolism is reflected in the presence of two distinct metabolic phases-a light-dependent anabolic phase tailored around the synthesis of storage compounds and metabolic precursors and a light-absent catabolic period that metabolizes the previously manufactured compounds to release energy in the absence of an external energy source. Due to these considerations, the analysis of phototrophic growth using constraint-based optimization methods is insufficient and needs to be extended beyond time-invariant descriptions. Here, we introduce CycleSyn which models the periodic nature of metabolism in Synechocystis sp. PCC 6803. Our approach enables us to account for temporal metabolic shifts tailored around light availability while still allowing for the use of the pseudo steady-state assumption used in conventional flux balance analysis. This is achieved by exploiting the large difference in time-scales between metabolic reactions and cell growth. We first validate the biological fidelity of CycleSyn predictions by comparing them to experimental observations for a diurnally cultured Synechocystis sp. PCC 6803 and to identify the major temporal variations in its metabolic processes. Next, we demonstrate the ability of CycleSyn to describe a temporally-varying metabolism by introducing diazotrophy in Synechocystis and evaluating the genes that need to be upregulated/downregulated to enable nitrogen fixation in a photosynthetic organism. Our study lays the foundation for subsequent analysis of systems with temporal variations in metabolism using a constraint-based optimization approach. | 12,749 | 380 | lay_plos | en |
Summarize: Get the latest from TODAY Sign up for our newsletter One Conifer, Colorado, couple says they narrowly escaped unspeakable tragedy after rescuing their 3-year-old daughter from inside their washing machine. Mom Lindsey McIver posted the story to Facebook, saying it's a cautionary tale to parents and caretakers about the dangers washer and dryers pose to children. “On Monday my husband went to Lowe’s and purchased this new front load washing machine,” McIver wrote. “We thought it was the ‘new and cool’ type of washing machine and didn’t think anything of it. We spent that evening installing it with the kids underfoot. We told them several times that they were not to touch it. They all replied ‘OK.’” Less than a day later though, everything changed. McIver and her husband said they awoke to their 4-year-old son crying so hard he could barely talk. “As I was trying to understand what he was saying, my husband flew out of bed and down the stairs. It was then that the realization hit. He had said: ‘Kloe. Inside. Washer,’” she recounted. Their young daughter was locked inside the washing machine with the machine rotating and water filling up inside. “We were able to quickly stop it and unlock the door and get her out. Aside from a couple of small bumps on her head and wet clothes, she was fine. After going through all the ‘what if‘s’ and ‘could have’s’ we know we are very blessed and God had mercy on our sweet daughter,” she wrote. Now she wants to educate other parents and caretakers about keeping your kids safe around front-load washers and dryers. She’s since installed a child safety lock on the machine and encourages others to take similar measures. To protect your children from washer and dryer entrapment, experts suggest engaging any appliance safety features, locking the door to the room where you keep appliances and purchasing a safety lock as well as talking to your kids about the dangers, among other measures. Getty Images “I took this picture after we secured the door shut with a child safety lock. We also found a child lock feature on the settings that, as long as it is engaged, will not allow the washing machine to start. But it does not lock the door,” she wrote. The maker of the machine, LG, responded, issuing a statement: “We applaud Ms. McIver for telling her story and share in her efforts to make sure that consumers are aware of the child safety lock feature available on LG washing machines and dryers. We encourage people to use this important safety setting and to contact our customer support team if they need any assistance.” The most terrifying part, McIver told NBC News, was that the door to the washer was locked. "My husband doesn’t remember what button he pushed," McIver said. "But I think he pushed pause and power, which helped us open the door. We believe what happened is that my daughter, Kloe, climbed inside and our son, who is 4, closed the door and pushed start. And then we think he panicked and came to get us." Their 6-year-old slept through the incident. While the kids are "still traumatized," the McIvers haven't brought the incident up with them again, beyond reiterating safety protocols. "They are still scared about what happened. They don't want to be in trouble." According to Taryn Brucia, Director of Public Relations for LG Electronics, the child safety lock that comes standard on all LG washers is not a requirement. "It's voluntary and optional," she told TODAY Parents via email. "LG has proactively elected to add this important safety feature to our products." She noted that LGE can't speak for other brands in the industry. "The child safety lock feature is available on all LG washers and models available at retailers nationwide," Brucia wrote. "On nearly every model, there is a Child Lock button on the control panel display. users should turn on the machine and then press and hold the Child Lock button for 5 seconds to activate it. Once activated, the machines cannot be used and a 'CL' (Child Lock) code will display." Parents who need assistance can find instructions in the user manual or call LGE customer support. More than 2,000 children each year are seriously hurt, and some die, after climbing, or falling into washers and dryers, or toppling down from them, according to Consumer Reports. The National Center on Early Childhood Health and Wellness encourages parents to keep their washers and dryers closed when not in use. But some young children can open the doors and climb in regardless. Watch: New gadgets that will keep your child safe at the beach or pool The Association of Home Appliance Manufacturers issued a statement: "Parents should feel comfortable talking with their children about safe and proper use of appliances in the home. U.S. safety standards address child entrapment, requiring that a latched door will open with 15 lbs. of force from the inside. However, once a clothes washer is activated, the door will lock and cannot be opened from the inside. Parents may also decide whether a third party latch on the outside of the washer is appropriate for their situation, depending on the age of the children in the home. We are heartened by the positive response to Ms. McIver’s Facebook post, and encourage all parents to be fully aware of the proper and safe use of their home appliances. It’s critically important to read the use and care manual to learn about available safety features and warnings." To be extra safe, the Consumer Protection Safety Commission suggests installing a lock to the door of your utility room, “child-proofing” appliances and warning children to not play inside these appliances. To “child proof” your washer and dryer, the Wyoming Montana Safety Council encourages parents to install dryer locks and locking straps. Mom whose toddler was locked in filling washing machine: 'We were sick to our stomachs' The McIver's added their own child safety lock to the washing machine where their toddler was locked. (Photo: Jennifer McIver) The Colorado family whose toddler was locked in a front-loading washing machine as it filled with water shared more details of their harrowing ordeal with USA TODAY on Monday and said they plan to contact the machine's manufacturer and return it for a top loader. "We don't want to slander the company," mother Jennifer McIver told All the Moms. "But at some point, we'd like to propose they add a feature so the door locks." LG, the company that makes the machine, did not respond when asked about adding a door lock but in a release to All the Moms pointed to a feature that prevents the door from being opened once a button is pushed and the machine is started. McIver wants a permanent lock on the door before the washing machine is started. "We applaud Ms. McIver for telling her story and share in her efforts to make sure that consumers are aware of the child safety lock feature available on LG washing machines and dryers. We encourage people to use this important safety setting and to contact our customer support team if they need any assistance. (LG customer support can be reached 1 (800) 243-0000)." 'She was screaming' On Monday, McIver revealed more details of the horrifying experience. She originally shared the story on Facebook. It has since gone viral with more than 266,000 shares. McIver said she was awakened by her crying 4-year-old around 7 a.m. Her children usually wake up before their parents, play a while and then come get them. Father Alan McIver caught the words, "Kloe, inside, washer," and ran to the basement. McIver guessed Kloe, 3, was in the washer for about two minutes. And she escaped with just some bumps to the head. "We think that our 4-year-old would have only had time to shut the door behind him, push start and then come right to us," she said, adding that the washer had maybe 2 cups of water in it. "Not enough that she could have drowned," McIver said, but she added that her daughter was tumbling around and was clearly terrified. "She was screaming. It was airtight, and it was soundproof. But you could see it and hear these muffled sounds as you stood right next to it." Jennifer McIver said she was originally hesitant to share her story for fear of mom-shaming, but she's managed to avoid that by avoiding comments on her Facebook post. She stands by her closing thoughts on the post that "we need to be open and honest about our mistakes to help one another keep our kids safe." Taking the machine back The McIvers, of Conifer, Colorado, plan on exchanging the front-loading machine they got at Lowe's for a top loader. While the McIvers added their own external child-safety lock to the front of the washing machine, Jennifer McIver said she would rather not look at it sitting in the basement after what happened to her daughter. "We were sick to our stomachs for the rest of the day — a couple of days, actually," she said. Like All the Moms? Follow us on Facebook and Twitter. READ MORE: Read or Share this story: https://usat.ly/2uyBZ9B | Summary: What seemed like a "new and cool" home upgrade nearly turned into a tragedy for one family in Conifer, Colo., and mom Lindsey McIver is now speaking out about it. In a Facebook post last Wednesday, McIver writes that she was somewhat reluctant to share her story due to fears of "the inevitable online mom-shaming," but that she felt like she needed to share "the danger of this machine"-her brand-new washing machine. On July 9 the family replaced their broken washer with a front-load model. The next morning the couple awoke to their 4-year-old son's cries. The words he managed to get out: "Kloe. Inside. Washer." They raced downstairs to find their 3-year-old daughter locked inside the airtight machine. There was roughly a pint of water inside and it was tumbling. The girl's screams weren't audible outside, and McIver believes she had been in the machine for about two minutes. They were able to stop it, their daughter emerged unharmed, and they installed a child safety lock on the door-something McIver pushes others to do. She subsequently discovered the LG machine has a child-lock setting that, when activated, prevents the washer from being turned on, but it doesn't keep the door in a locked mode. "We don't want to slander the company," she tells USA Today. "But at some point, we'd like to propose they add [that] feature." She's not going to wait for it: The McIvers plan to swap the machine for a top loader. In a statement, LG "applaud[ed]" McIver for sharing her story. A rep for LG tells the Today Show the child lock feature it offers isn't required by law, but that "LG has proactively elected to add" it to its products. | 2,113 | 428 | multi_news | en |
Summarize: RELATED APPLICATIONS This Patent Application is a continuation application of U.S. patent application Ser. No. 10/639,962, filed Aug. 12, 2003, and entitled, “JUMP ROPE SIMULATOR,” now U.S. Pat. No. 7,172,534, which claims priority under 35 U.S.C. 119(e) of the U.S. Provisional Patent Application, Ser. No. 60/403,749, filed Aug. 13, 2002, and entitled, “JUMP ROPE SIMULATOR AND METHOD OF EXERCISE.” The Provisional Patent Application, Ser. No. 60/403,749, filed Aug. 13, 2002, and entitled, “JUMP ROPE SIMULATOR AND METHOD OF EXERCISE” and U.S. patent application Ser. No. 10/639,962, filed Aug. 12, 2003, and entitled, “JUMP ROPE SIMULATOR” are both is also hereby incorporated by reference. FIELD OF THE INVENTION The present invention relates generally to aerobic and anaerobic exercise devices, and more specifically to a new jump rope simulator for providing aerobic and anaerobic exercise. BACKGROUND OF THE INVENTION Traditional jump ropes are usually made from a single length of rope with handles at both ends for the user to grip. The user holds the rope by the handles, swings the rope over his head and then under his feet in a continuous motion, and jumps over the rope every time it passes under his feet. If the user should misjudge when the rope is under his feet, he will trip over the rope and lose the rhythm and timing of the exercise workout. One way to obtain the beneficial aerobic workout associated with a traditional jump rope and yet avoid tripping over a rope is to use a jump rope simulator which does not actually pass a rope under the user's feet. One hand-held unit or two unconnected hand-held units are grasped in the user's hands and rotated while the user skips periodically and rhythmically, without having to actually jump over a physical rope, thus simulating a traditional jump rope exercise. The use of jump rope simulators for aerobic exercise is known in the prior art. U.S. Pat. No. 5,895,341 to Jones entitled, “Jump Rope Simulator,” discloses a pair of hand-held units each having a handle and a flexible cord with a weight distribution biased toward the free end of the cord and intended to be rotated about an axis extending from the handle in a manner similar to the motions employed when using a standard jump rope. Optional embodiments include handles that contain a battery-powered jump counting device with display and a battery-powered calorie counting device with display. U.S. Pat. No. 6,524,226 to Kushner entitled, “Exercise Device,” discloses a pair of elastic bands each having a longitudinal handle and a lateral handle. The elastic bands can be joined together with a fastener and used as a single resistive force device for isometric exercises, or the two elastic band units can be held individually, one in each hand of the user, for use as a jump rope simulator. The lengths of the elastic bands may be adjusted through the use of pins that are removably positioned in apertures located in the handle and band. While these patents disclose an exercise device wherein a user can simulate the motions associated with using a traditional style jump rope, neither of the disclosed constructions allow the user the option of using the jump rope simulator as an individual unit or as a linked device formed by connecting two individual units which results in a device similar in form and function to a conventional jump rope. SUMMARY OF THE INVENTION The present invention is a device and method to be used for aerobic and anaerobic exercise. A jump rope simulator of the present invention has a hand-held unit comprising a handle, a fixed length tube with one end permanently attached to the handle, a series of concatenated tubular beads removably connected to the second end of the tube, and a ball-shaped safety nodule attached to the bead farthest removed from the tube. A simple mechanism is used for connecting the beads together so that a user may quickly adjust the length of the unit. The length of the unit can be repeatedly adjusted by adding or removing beads from the unit. Preferably the length of the unit is adjusted such that the safety nodule just touches the floor when the handle of the unit is held in a relaxed position by the user's side. A sound mechanism is preferably included in the handle so that as the fixed length tube rotates around the hand-held unit, a sound is made on each rotation. A connector is used to link together two units of the jump rope simulator by removing the safety nodules and linking the beads farthest removed from the handles of the two units. A simple mechanism is also used to link the connector to the last bead of the two units so that a user is able to quickly convert between the two configurations of the jump rope simulator. An alternative embodiment includes a flexible cord which runs down the center of the tube and beads and is attached to the handle on one end and the safety nodule or the last bead farthest removed from the handle on the other end. The presence of the cord in the unit ensures that the beads and the safety nodule are secured to the handle. Even if one of the connections were to inadvertently release during an exercise workout, none of the pieces would fly off, and the jump rope simulator would still function. Having this safety feature would be especially important in a group exercise environment such as a fitness club or an aerobic and/or anaerobic workout class. According to an embodiment of the present invention, the beads are attached to each other through the use of a simple, low-cost interlocking snap-fit mechanism on the beads. One end of each bead has a spherical protrusion and the other end of each bead has a socket that accepts the spherical protrusion. The first end of one bead plugs into the second end of another bead, and the beads can be easily added or removed by a user without the use of any tools. In this way, a user can adjust the number of beads on the unit and thus adjust the length of the unit to accommodate the user's height. Preferably the bodies of the tubular beads have accordion-like pleated folds to provide for flexibility in the unit so that the unit moves freely and mimics the feel of a rope during use and is also easy to store and transport between uses. According to another embodiment of the present invention, the beads are attached to each other with the use of threaded screw-type male and female ends on the beads. One end of each bead is a threaded male end and the other end of each bead is a threaded female socket. The first end of one bead screws into the second end of another bead, and the beads can be easily added or removed by a user without the use of any tools. In this way, a user can adjust the number of beads on the unit and thus adjust the length of the unit to accommodate the user's height. Preferably the bodies of the tubular beads have accordion-like pleated folds to provide for flexibility in the unit so that the unit moves freely and mimics the feel of a rope during use and is also easy to store and transport between uses. According to yet another embodiment of the present invention, the beads are attached to each other through the use of a screw-type coupler between the beads. Both ends of each bead are threaded female sockets. A tubular coupling screw having two threaded male ends is used to easily link together two beads. Alternatively, both ends of each bead are threaded male ends, and a tubular coupling screw having two threaded female ends is used to easily link together two beads. This simple low-cost connecting mechanism between the beads allows the user to adjust the number of beads on the unit and thus to adjust the length of the unit to accommodate the user's height without using any tools. Preferably the body of the tubular coupling screw has accordion-like pleated folds to provide for flexibility in the unit so that the unit moves freely and mimics the feel of a rope during use and is easy to store and transport between uses. Incorporating elements containing accordion-like pleated folds into the units of the jump rope simulator not only allows units to bend but also to stretch. Units having this versatility are used effectively, either as individual units or as linked units, for a variety of stretching or Pilates-type movements for body conditioning or physical therapy programs. Optional features of the present invention include different safety nodules having various weights which serve to increase the resistance felt by the user as the unit is being rotated or swung in the user's hand, a light emitting device such that the safety nodule at the end of the unit emits light as the user is exercising and alerts other people in the neighboring vicinity that the unit is in motion, a two-piece rotatable ergonomic handle with a right-angle bend that helps to guide the safety nodule end of the unit away from the user's legs when the unit is being rotated, and a variable weight handle capable of holding weights to increase the resistance felt by the user as the unit is being rotated or swung in the user's hand. Preferably the variable weight handle is a hollow handle in which different valued weights may be loaded. Another alternative to varying the weight of the handle is to screw weights onto the free end of the handle. In such a configuration, the end-cap of the handle is removed, exposing a threaded female socket for accepting a screw-on weight. The screw-on weight is a disk that has the same diameter as the handle with a threaded male end on one side of the disk for screwing into the free end of the handle. The other side of the disk has a threaded female socket for accepting an additional screw-on weight or the handle's end-cap. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the preferred embodiment of the jump rope simulator in accordance with the present invention. FIG. 2 shows two units of the jump rope simulator linked together with a removable connector. FIG. 3 shows an exploded view of two beads and a safety nodule having a snap-fit type of connection according to an alternate embodiment of the present invention. FIG. 4 shows an exploded view of two beads and a safety nodule with a screw-coupler type of connector according to an alternate embodiment of the present invention. FIG. 5 shows an exploded view of the end of the handle and a screw-on weight according to an alternate embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION FIG. 1 illustrates a jump rope simulator 100 in accordance with the preferred embodiment of the present invention. The jump rope simulator unit 100 has a handle that is L-shaped and padded on the long side 101 with a resilient material in an ergonomic shape that fits comfortably in a user's hand during exercise workouts. The handle has a short side 103 that is attached to the long side of the handle 101 by a ball-bearing joint 102 which allows the short side of the handle 103 to rotate 360 degrees around the longitudinal axis of the long side of the handle. A sound mechanism 114 is preferably built into the ball-bearing joint 102 such that for each rotation of the handle 103 around the long side of the handle, an audible sound is output. The rotatable handle is designed so that when the user rotates the individual unit, the far end of the unit is prevented from impacting the user's legs. The long side of the handle 101 is preferably hollow and has a removable end-cap 120. A weight 121, available in various values, can be inserted inside the hollow handle. The weight adds extra resistance when the user is rotating the jump rope simulator in an aerobic and anaerobic workout. Also shown in FIG. 1 is a fixed length tube 104 permanently attached to the end of the short side of the handle 103. The tube provides stability to the unit as it is being rotated by the user and also contributes to guiding the ends of the unit away from the user's legs in conjunction with the L-shaped handle. Along the body of the tube 104, there are alternating sections that are smooth 105 and accordion-like with pleated folds 106. The pleated folds provide the tube with some flexibility in bending when a user is rotating the jump rope simulator and also allow bending of the units so that the unit can be stored in a compact manner. Attached to the bottom of the tube 104 is a sequence of several beads that are each preferably shorter in length than the tube 104. Only two beads are shown in FIG. 1, although any appropriate number of beads may be connected by the user to adjust the overall length of the unit to reach the floor and accommodate the user's height. The bead 108 is connected to the tube 104 at the connector 107, while the bead 110 is connected to the bead 108 at the connector 109. Each of the beads 108 and 110 also have accordion-like pleated folds in the body to provide for flexibility in the bead. At the bottom of the last bead on the unit is a ball-shaped safety nodule 112 which is attached to the last bead 110 by the connector 111. The round safety nodule 112 is preferably made out of a soft material so that if the user inadvertently hits himself or another person while using the jump rope simulator, the safety nodule will cushion the impact. The safety nodule 112 is available in several different weights that provide variable resistance to the user during his aerobic and anaerobic workout with the jump rope simulator. In some embodiments, the safety nodule 112 further comprises a light emitting device 113 such that the safety nodule 112 at the end of the unit emits light as the user is exercising and alerts other people in the neighboring vicinity that the unit is in motion. As used herein, the term beads refers to any non-locking or interlocking segments, sections, tubular elements, collars or cylinders which can be used to extend the length of the jump rope simulator of the present invention. The length of the unit can be repeatedly adjusted by adding or removing beads from the unit. This allows the same unit to be repeatedly adjusted for use by users of different heights. Conventional jump ropes typically only allow for a single length adjustment by cutting the rope. FIG. 2 illustrates two units 202 and 203 of the jump rope simulator linked to each other with a removable connector 201. In this configuration, the safety nodule 112 at the end of the unit shown in FIG. 1 is removed from both of the units 202 and 203, and the connector 201 is directly connected to the bead farthest away from the handle of each unit. The linking of the two units results in a device 200 that can be used in a similar manner as a conventional jump rope. The connector 201 is available in several different lengths to allow the user to modify the total length of the linked jump rope simulator to fit a particular user's height. The connector 201 can be positioned anywhere between the two handles of each unit. FIG. 3 is an exploded view of two beads 310 and 320 and a safety nodule 330 attachable to each other with a snap-fit type of connection. The two beads 310 and 320 each have a spherically-shaped protrusion 311 and 321 on one end and a socket 312 and 322 for accepting a spherically-shaped protrusion on the other end. The tubular body of each bead 313 and 323 has accordion-like pleated folds which allow for flexibility in bending the bead. As shown in FIG. 3, the spherically-shaped protrusion 321 on the top end of the bottom bead 320 plugs into the socket 312 in the bottom end of the top bead 310 in a snap-fit manner. This simple mechanism allows users to easily add or remove beads to adjust the length of the unit to accommodate the user's height. Plugged into the socket 322 located at the bottom end of the bottom bead 320 is a ball-shaped safety nodule 330 which is made out of a soft material. The safety nodule 330 also has a spherically-shaped protrusion 331 for plugging into the socket 322. In addition the exploded view in FIG. 3 shows a flexible cord 340 on which the beads are strung; in ordinary use, after assembly the cord would be hidden from view. The flexible cord 340 ends in a knot 341 which securely fastens the safety nodule 330 to the unit. FIG. 4 is an exploded view 400 of two beads 410 and 420 and a safety nodule 440 attachable to each other with a screw-coupler type of connector. Both ends of each of the beads 411, 412, 421, and 422 is a threaded female socket. A coupler screw 430 having two threaded male ends 431 and 432 is used to connect the two beads 410 and 420. The tubular body 433 of the coupler screw 430 has accordion-like pleated folds to allow the connected beads to have flexibility in bending. This simple screw-type mechanism allows users to easily add or remove beads 413 and 423 to adjust the length of the unit to accommodate the user's height. As shown in FIG. 4, the safety nodule 440 has a ball shape 441 and a threaded male end 442. The threaded male end 442 of the safety nodule screws into the bottom 422 of the last bead in the series. FIG. 5 is an exploded view 500 of the free end of the handle 510 and a screw-on weight 520. The free end of the handle 510 has a threaded female socket 511 to accept the screw-on weight 520. The screw-on weight 520 is disk-shaped and has a threaded male end 521 on one side which screws to the free end of the handle 510. The other side of the screw-on disk-shaped weight has a threaded female socket 522. This socket can accept another screw-on weight to increase the total weight of the handle or the handle end-cap 530 which also has a threaded male end. To assemble a jump rope simulator, a user takes the handle with attached tube and attaches a bead to the free end of the tube. The user continues attaching additional beads to increase the total length of the unit until a length appropriate for the user is reached. The ideal length allows the safety nodule to just touch the floor when the handle of the unit is held in a relaxed position by the user's side. Finally, at the end of the concatenation of beads the user attaches a safety nodule. Use of the jump rope simulator as an exercise device is straightforward. For the configuration where the device is used as an individual unit or a pair of units, as shown in FIG. 1, the user simply grasps the handle in his hand and rotates the unit(s), simulating the feeling and rhythm of rotating a conventional jump rope. Because of the sound mechanism 114 within the ball-bearing joint 102, the user also receives a simulated audible feedback as if a jump rope were hitting the floor on each rotation. The user can also jump or skip on each rotation of the rotating handle and exercise continuously without worrying about tripping over an actual rope and losing his balance, rhythm, and timing as would typically occur when a user trips over a conventional jump rope. However, should the user desire to exercise with a traditional style jump rope, two units of the simulator are easily connected together as shown in FIG. 2. The connection and detachment of the two units is very simple, thus allowing the user to choose which exercise device configuration meets his needs and to quickly switch between the two configurations as desired. Multiple users have the ability to each utilize a pair of jump rope simulators in a group or class type environment in order to participate in a group workout. This allows the group of users to perform a series of exercises using the jump rope simulators of the present invention for aerobic and anaerobic exercise. This series of exercises can be performed to music to provide a rhythm and pace for the exercise. Further, the level of exertion of the user's exercise program can be changed by increasing or decreasing the amount of weight loaded in the handle or attached to the end of the handle. Alternatively, the safety nodule at the end of the unit may be exchanged for another safety nodule having a different weight. The weight of the unit can also be increased or decreased by any other appropriate manner, including adding weight to the handle or to the end of the beads or safety nodule. The present invention discloses an exercise device wherein a user can simulate the motions and aerobic and anaerobic exercise benefits associated with using a traditional style jump rope without the risk of tripping over a physical rope. The jump rope simulator of the present invention has several advantages over the prior art. A user is able to easily and repeatedly modify the length of the jump rope simulator by adding or removing beads. A user is also able to easily and repeatedly modify the weight of the jump rope simulator either at the handle or at the safety nodule in order to increase or decrease the resistance of the unit as it is being rotated in the user's hand. Also, if the user desires, two of the jump rope simulator units are easily linked together with a simple connector to form a device similar to a conventional jump rope in form and function. The teachings of the present invention allow a user to quickly convert between the two configurations of the jump simulator. The present invention has been described in terms of specific embodiments incorporating details to facilitate the understanding of the principles of construction and operation of the invention. Such reference herein to specific embodiments and details thereof is not intended to limit the scope of the claims appended hereto. It will be apparent to those skilled in the art that modifications can be made in the embodiment chosen for illustration without departing from the spirit and scope of the invention. Specifically, it will be apparent to one of ordinary skill in the art that the device of the present invention could be implemented in several different ways and have several different appearances. | Summary: A jump rope simulator for aerobic and anaerobic exercise having a hand-held unit comprising a handle, a base length connected to the handle, and one or more concatenated extension lengths attached to the base length. To exercise, the user grasps one or two units, imitates the motions of swinging a conventional jump rope, and jumps up and down, but there is no risk to the user of tripping over a rope as with a conventional jump rope. If the user desires, two units may also be linked together with an easily attachable connector to form a device similar to a conventional jump rope. A user can quickly convert between the two configurations of the jump rope simulator. | 5,361 | 152 | big_patent | en |
Summarize: [0001] This application claims priority of Provisional Application Ser. No. 60/560218 filed Apr. 18, 2004. FIELD OF THE INVENTION [0002] The present invention relates to a gel composition for use in reducing the risk of infection caused by sexually transmitted viruses, such as herpesviruses (herpes simplex, cytomegalovirus and the like), influenza A, parainfluenza, hepatitis B, human papilloma viruses, and in particular human immunodeficiency virus (HIV). The present invention also relates to a method for reducing the risk of infection by the above identified viruses as a result of sexual intercourse by the use of the gel composition during sexual intercourse. BACKGROUND OF THE INVENTION [0003] Acquired human immunodeficiency syndrome (commonly abbreviated as AIDS) is a disease caused by human T-cells becoming infected by a causative virus present in human blood. The virus is known as acquired human immunodeficiency syndrome virus which commonly is abbreviated as HIV. [0004] The spread of HIV worldwide has caused significant loss in both human lives and in the economy, especially in third world countries such as sub-Saharan Africa and, increasingly, in some Asian countries. The most promising drugs developed and currently under development include, for instance, azidothymidine (AZT), bis-deoxycytidine, suramine, phosphonoformate, antimony tungstate, papaverine as disclosed in U.S. Pat. No. 4,971,977, 2′,3′-dideoxyinosine as disclosed in U.S. Pat. No. 5,026,687, benanomicins A and B as disclosed in U.S. Pat. No. 5,120,717, porphyrin as disclosed in U.S. Pat. No. 5,192,788, and inhibitory peptides as disclosed in U.S. Pat. No. 4,880,779. One major limitation of these drugs and many others is that they are typically highly toxic. Another important limitation is that these drugs are very expensive and therefore are not affordable by the economically disadvantaged patients in the developing countries where HIV infections strike hardest. One further limitation is that the regimen for their use is typically tedious and hard to follow. However non-adherence to the regimen can compromise the efficiency of the drug and also enhance the possibility of resistance to the drug by the virus. [0005] It has been concluded that the most effective way to control the spread of HIV infection and AIDS disease is to prevent the infection of a healthy person by that virus. [0006] HIV can be transmitted from an infected person to a healthy person when the healthy person comes into contact with the blood, semen, vaginal fluids, and breast milk from the infected person. The four general modes of HW transmission include: unprotected sex, blood contact, the use of HIV virus contaminated syringes and/or needles, and mother to child before, during and after childbirth. A major method of HIV transmission could take place during unprotected sexual intercourse due to the exchange of bodily fluid. Therefore, AIDS is regarded as a venereal disease with a fatal outcome, and in as many as 80% of the world's diagnosed cases, is caused by the passage of HIV across the genital mucosa (U.S. Pat. No. 5,552,383 and Merson, Science, 260: 1266-8, 1993). While condoms can be used to reduce the likelihood of transmission of HIV virus during sexual contacts, the use of condoms is not always strictly adhered to due to various reasons, including their cost, religious prohibition, and the diminished sexual satisfaction. [0007] A method which is convenient and easily implemented, for reducing the risk of contracting HIV and the other sexually transmitted viruses, during sexual intercourse is by the use of a pharmaceutical composition, in the form of gel, lubricant cream or the like, by at least one of the individuals involved. Clinical surveys have suggested that the use of spermicides with known antiviral activity, e.g. nonoxynol-9, will not satisfactorily restrict the spread of HIV (Cates et al. Family Planning Perspective, 24:75-84, 1992), as the frequent use of nonoxynol-9 leads to the creation of vaginal lesions (genital ulcers and vulvitis), which provide entry ports for HIV particles to pass through the protective barrier of the skin and reach the bloodstream (Kreiss et al. The Journal of The American Medical Association, 268: 477-482, 1992). Kelly in U.S. Pat. No. 5,624,675 disclosed a method of using a topical genital lubricant with a non-toxic, non-irritating zinc salt during sexual intercourse for the reduction of HIV infection. The mechanisms are such that 1) zinc can promote healing and closure of lesions, microabrasions, and other skin breaches and thereby reduces the ability of HIV to penetrate the skin and reach lymphocytes, 2) zinc ions form crosslinking bonds with cysteine and histidine residues in the HIV protein and thereby attach HIV particles to each other, to proteins in vaginal fluids, and to dead or dying cells that will be soon discharged from the vaginal surface which reduces the ability of the HIV virus to infect the susceptible healthy cells, and 3) concentrations of zinc that do not harm the skin can kill HIV-infected lymphocytes and prevent the lymphocytes from further infecting other cells. Bourinbaiar in U.S. Pat. No. 5,552,382 disclosed the use of gramicidin as an active ingredient of spermicide with virucidal activity against HIV. Shihata in U.S. Pat. No. 5,778,886 disclosed the use of nonoxynol-9 and hydrogen peroxide in vaginal compositions for the prevention of conception and the transmission of sexually transmitted disease. Porat in U.S. Pat. No. 6,624,198 disclosed a spermicidal lubricant composition, which includes chlorhexidine salt as an active ingredient against HIV and other viruses. SUMMARY OF THE INVENTION [0008] In accordance with the invention it has now been found that the use of a specially formulated gel as a lubricant during sexual intercourse, which contains, as an active ingredient, benzoic acid in a suitable pharmaceutical carrier, can significantly de-vitalize the HIV virus, and/or other sexually transmitted disease viruses upon contact within a reasonable period of time. The gel is also capable of serving as a contraceptive by devitalizing the sperm. [0009] It is a primary object of this invention to provide a composition for the prevention of the transmission of HIV and other sexually transmitted disease viruses during sexual intercourse. [0010] It is a primary object of this invention to provide a method for the prevention of the transmission of HIV and other sexually transmitted disease viruses during sexual intercourse. [0011] It is another object of this invention to provide a method for utilizing benzoic acid in a suitable formulation that can be spread on the genitals prior to or during sexual intercourse to reduce the risk that uninfected person will become infected by HIV and other viruses. [0012] Another object of this invention is to provide a sexual lubricant, gel, or cream which contains an effective topically-active anti-HIV agent which is non-toxic and non-irritating to the genitals and urethral and vaginal membranes. [0013] The compositions can be applied to the external genitalia as well as internal mucosal surface to prevent transmission of HIV and other sexually transmitted disease viruses. [0014] Yet another object of this invention is to provide a sexual lubricant, gel, or cream which contains an effective topically-active anti-HIV agent, which is non-toxic and non-irritating to the genitals and urethral and vaginal membranes and which can be used in conjunction with a prophylactic device, such as a condom or a diaphragm. DETAILED DESCRIPTION OF THE INVENTION [0015] In order to maintain the activity of benzoic acid against HIV and other sexually transmitted disease viruses, it is essential to maintain the pH value of the composition at or below 4.0. The pH of a healthy vagina is also mildly acidic (pH 3.5-4.5) and this acidity is generated by the production of lactic acid by lactobacilli, which form a major component of the healthy vaginal flora. Together with other factors, the acid pH is recognized as an essential part of the protective mechanisms against infections. While the acidic conditions can inactivate sperms and viruses, semen contains a potent alkaline buffering capacity that neutralizes the vaginal acidity for a period of many hours after intercourse, therefore diminishing the virus inhibiting capacity of the acids (Lezdey et al in U.S. Pat. No. 6,428,791). [0016] Regular gels do not perform well under such acidic conditions because the acidity tends to destabilize the gels. In accordance with the invention a special gel is provided, “Origen”, that can be used in acidic conditions as exist in the vagina and surrounding area without destabilization. The gel is made by the co-polymerization of polyquaternium-4 and polyacrylamidomethylpropane sulfonic acid in a pharmaceutically acceptable carrier. A preferred carrier for this purpose is a water base consisting about 60% water and about 20% glycerin. The presence of the glycerin is important in that it increases the spread of the gel on the surface of genitals. The lubricity of the gel further helps reduce the likelihood of transmitting the diseases by reducing the rupture of skins and/or blood vessels during sexual relationships. The gel of the invention is activated by being made alkaline with sodium hydroxide to produce a uniform co-polymerization and solubilization, then acidified with hydrochloric acid to reach the desire acid pH of about 3.8. [0017] Polyquaternium-4, commercially available from companies such as National Starch under the trade name CELQUAT® L-200 or H-100, has been used primarily in the skin and hair care industry in formulations in conditioners, cream rinses, creams, detangling spritzes, liquid soaps, mousses, setting lotions and skin lotions. Polyquaternium-4 is a cationic, polymeric, and water soluble quaternary cellulose derivative have the following structure. [0018] Polyacrylamidomethylpropane sulfonic acid, whose structure is shown below, has theretofore been used as a film former, a thickener, a lubricity additive and a moisturizer for personal care and home care applications (U.S. Pat. No. 4,128,631). It is commercially available, for example, under the trade name COSMEDIA® HSP-1180 by Cognis. [0019] To the gel with the co-polymerized polyquaternium-4 and polyacrylamidomethylpropane sulfonic acid in the above-mentioned water base, a preservative can be added. A preferred preservative is selected from one or more type of parabens. The parabens are a group of closely related chemicals which are esters of p-hydroxybenzoic acid. They are used widely as preservatives for cosmetics, foods and drugs. Other known preservatives or combinations thereof can also be used instead, especially for the formulations prepared for the individuals that are allergic to parabens. The active, benzoic acid should be added at a concentration generally of about 0.2% to no more than 2%, as too great an amount will inhibit the normal and necessary flora in the vaginal tract. The benzoic acid further acts as a stabilizer to prevent a fall off of activity if the product should become diluted. [0020] The following preferred formulation of the gel of this invention is provided as an example, Ingredient Amount (% by wt) Polyquaternium-4 0.70 Polyacrylamidomethylpropane 3.30 sulfonic acid Water QS to 100% Glycerin 10.00 Sodium hydroxide 0.15 Hydrochloric acid QS to pH 3.8 Benzoic acid 0.30 Methylparaben 0.20 [0021] In order to evaluate the efficacy of the Origen gel in inhibiting HIV viruses in both concentrated and diluted forms, the following tests were conducted. [0022] Four sets of samples were prepared according to the following steps which are test samples based on Origen gel at a first series of dilutions, control samples based on AZT at a second series of dilutions, a positive control sample and a negative control sample. [0023] Step 1. For the Origen samples, Origen gel was diluted in sterile distilled water giving rise to dilutions ranging from 1:5 to 1:500,000 (2× concentrations). For the AZT samples, an AZT stock was diluted in H9 media giving rise to dilutions whose concentrations were 2, 0.2 and 0.02 μM (2× concentrations). The H9 cells are a type of HIV-1 permissive human T-cell lymphoma. Typical H9 media comprise RPMI, fetal bovine serum (FBS), penicillin, streptomycin and glutamine, for example, RPMI 1640 supplemented with 20% fetal bovine serum, 2 mm 1-glutamine, 100 U·mL −1 penicillin, and 100 μg·mL −1 streptomycin, and 5% CO 2. [0024] Step 2. For each of the Origen samples, an aliquot of 50 μl Origen gel dilution was mixed with 50 μl HIV-1 (500 TCID 50 ) in a 1.2 milliliter centrifuge tube by vortexing to provide final Origen gel dilutions (1:10 to 1:1,000,000). For each of the AZT samples and the positive control sample, 50 μl water was mixed with 50 μl HIV-1 in a 1.2 milliliter centrifuge tube by vortexing. For the negative control sample, 100 μl of water was added to a 1.2 milliliter centrifuge tube. [0025] Step 3. The sample tubes were incubated for 15 minutes at 37° C. and 5% humidity. [0026] Step 4. An aliquot of 900 μl media for H9 cell was added to each tube. The tubes were centrifuged at 17,000 rpm at 4° C. for 90 minutes to pellet the viruses. The supernatants were discarded. [0027] Step 5. For the Origen samples and the positive control sample, each pellet was re-suspended in 500 μl H9 media by vortexing. For the AZT samples, each pellet was re-suspended in 500 μl of 2× AZT dilution by vortexing. [0028] Step 6. An aliquot of 100 μl sample was transferred from each tube into quadruplicate wells in a 96-well flat-bottomed plate. [0029] Step 7. An aliquot of 100 μl H9 cells (diluted to 1×10 6 cells/ml in H9 media) were added to each well, giving rise to a final concentration of H9 cells of 5×10 5 /ml. The AZT control samples have final concentrations of 1, 0.1, 0.01 50 μM. [0030] Step 8. The samples were incubated for 3 days at 37° C. and 5% humidity. [0031] Step 9. On the beginning of the fourth day, the culture supernatant was removed and discarded. For the Origen samples, the positive control sample and the negative control sample, 100 μl fresh H9 media were added. For the AZT samples, 100 μl 1× AZT dilution was added. [0032] Step 10. The samples were further incubated for 3 more days at 37° C. and 5% humidity. [0033] Step 11. On the beginning of the seventh day, the H9 cells from the culture were collected for indirect immunofluorescence assay (IFA) to detect HIV-1 antibodies. [0034] Step 12. Viability of the H9 cells was checked using trypan blue exclusion method. [0035] The result of the test is summarized in the following table. Drug dilution % cell viability % IFA % inhibition Origen Gel 1:10 80% 0% 100% Origen Gel 1:100 80% 0% 100% Origen Gel 1:1000 80% 0% 100% AZT 1 μM 80% 0% 100% AZT 0.1 μM 80% 20% 77.8% AZT 0.01 μM 80% 80% 11.2% Positive control 80% 90% NA Negative control 90% 0% NA [0036] The results show that Origen gel is generally nontoxic and that it can completely inhibit the HIV viruses at dilutions as high as 1000 times. Therefore, the gel comprising the above-mentioned compositions is capable of destroying any HIV and/or other sexually transmitted viruses that come into contact therewith in the vaginal tract in a matter of hours to a maximum of one day. Due to the ability of benzoic acid for deactivating sperm, the gel provides a further advantage by serving as a contraceptive. The composition further possesses the following advantageous properties, 1) the composition is compatible with the conditions necessary to maintain a healthy vaginal condition, 2) the composition is of excellent stability and freedom from self-liquefaction in that body cavity that might cause the composition to run or leak from he cavity; 3) the composition continues to be effective even if significantly diluted by some bodily fluids; and 4) the composition can be produced for sale at costs low enough to be obtainable and usable by the affected people of the world, especially those with limited incomes. [0037] The gel can be directly applied to either the male or female genitalia (such as penis and/or vagina) or any other bodily cavities, such as the rectum, which might be involved in the sexual intercourse prior to the sexual intercourse. The gel can also be used as a lubricant in combination with a prophylactic device, such as a condom or a diaphragm. When a condom is used in conjunction with the gel, the gel is capable of enhancing the risk-reducing effectiveness of condom and providing maximum protection for users. Such an increase in the anti-viral protection offered by a condom becomes important in various situations, such as when a condom breaks or if the male loses his erection prior to withdrawal and spillage or leakage of fluid occurs from the inside of the condom into the vagina. The gel can be coated onto condoms during manufacture, and enclosed within conventional watertight plastic or foil packages that contain one condom per package, or it can be manually applied by a user to the inside and/or the outside of a condom, immediately before use. The Origen gel composition can also be used by doctors and in hospitals to lubricate and sanitize gloves or instruments before examining the penis, vagina, rectum and the like. [0038] During sexual intercourse, the active in the gel, benzoic acid, deactivates the viruses in a matter of several hours to a maximum of one day and prevents the infected person from transmitting sexually transmitted disease viruses to the non-infected person. The ability of the gel to maintain its efficacy up to 1000× dilution is of paramount importance for the intended protection due to the secretion of bodily fluids inside the bodily cavities which dilutes the gel during sexual intercourse. The gel provides lubricity which further helps reduce the likelihood of transmitting the diseases by reducing the rupture of skins and/or blood vessels during sexual relationships. [0039] The Origen gel can be packaged in any of the known forms, such in tubes, in bottles, in capsules or as an impregnant for treated towlettes which can be used to wipe the sex organs. | Summary: A gel composition and methods for its use singly or in combination with prophylactic devices in safe sexual relations, including prevention of infection by HIV and other sexually transmitted viruses. The composition contains benzoic acid as the active ingredient in an amount up to 2%, a copolymer of polyquaternium-4 and polyacrylamidomethylpropane sulfonic acid and a pharmaceutically acceptable carrier such as a water base consisting of about 60% water and about 20% glycerin. The composition may further contain a preservative, such as one or more members of parabens. The gel can maintain its antiviral activity in dilutions of not less than 1000 times. The methods for preventing the transmission of HIV and other sexually transmitted viruses comprise applying the gel composition directly onto genitalia or intravaginally before and/or during sexual intercourse or using the gel composition in combination with a prophylactic device, such as a condom or a diaphragm. | 4,833 | 212 | big_patent | en |
Summarize: Story highlights Donald Trump claimed Sunday that the 9/11 attacks wouldn't have happened on his watch Trump again attacked Jeb Bush over his brother's record on security Washington (CNN) Donald Trump says he's not blaming George W. Bush for 9/11, but he claims that if he'd been president, the attacks never would have happened. In an appearance on "Fox News Sunday," the real estate mogul said that since he's "extremely, extremely tough on immigration" the attackers wouldn't have been in position to commandeer U.S. flights. "So there's a good chance that those people would not have been in our country," Trump said. He took another shot at Jeb Bush for claiming that Bush's brother, the 43rd president, kept the nation safe. "I'm not blaming George Bush," Trump said. "But I don't want Jeb Bush to say, 'My brother kept us safe,' because September 11 was one of the worst days in the history of this country." Read More Ben Carson doesn’t believe Donald Trump was blaming former President George W. Bush for the Sept. 11th attacks in comments he made last week. Interested in? Add as an interest to stay up to date on the latest news, video, and analysis from ABC News. Add Interest During an interview with Bloomberg, Trump appeared to criticize Bush for the terror attacks. Carson told ABC's "This Week" today he doubted that was really what the real estate mogul meant. “I would probably ask him what he meant by that. I seriously doubt that he’s saying that -- that George W. Bush is to blame for it,” Carson said. "I certainly -- I certainly don’t think so.” Bloomberg’s Stephanie Ruhle had asked if Trump could reassure Americans in times of national emergencies, as the 43rd president did after Sept. 11. “I think I have a bigger heart than all of them," Trump responded. "I think I’m much more competent than all of them. I mean, say what you want, the World Trade Center came down during his time.” “Hold on,” Ruhle interjected. “You can’t blame George Bush for that." “He was president, OK? Don’t blame him or don’t blame him, but he was president," Trump continued. "The World Trade Center came down during his reign. If you look at Sandy Hook, those people are still begging for help." Carson, who is neck-and-neck with Trump in the latest GOP polls, was careful not to compare himself to his rival, who often highlights his negotiating prowess. But the retired neurosurgeon said he is also an experienced negotiator. “I don’t want to necessarily compare myself with anyone, but I can tell you that, you know, I’ve had a lot of experience doing a whole host of things -- negotiating with all kinds of people in order to get things accomplished,” Carson said. “When you go into a negotiating, the recent Iran negotiation, for instance, you have to know how to negotiate.” "For instance, when I became the director of pediatric neurosurgery at Johns Hopkins, pediatric neurosurgery was not even on the map at Hopkins at that point," he added when asked for specific examples. "I had to negotiate a number of things in order to create the various different divisions.” Carson also said he doesn’t think any of his potential Democratic opponents would be tough to beat in the general election. "I personally don't think any of them will be very tough because it's going to be such a clear-cut election," he said. "We will be voting about whether we want a nation where the government is in control or a nation where the people are in control. I think it's going to be crystal clear and the people will make a clear decision." Carson's campaign manager has claimed he never viewed Hillary Clinton as a threat but admitted he was concerned about the potential of Vice President Joe Biden entering the race. “Biden worries us a little more because he has the likability factor,” campaign manager Barry Bennet said last week. | Summary: Donald Trump continued his recent attacks on the Bush family by implying today that he would have prevented the 9/11 attacks, CNN reports. The Republican presidential contender said on Fox News Sunday that he's "extremely, extremely tough on immigration," which may have thwarted the 9/11 terrorists. "There's a good chance that those people would not have been in our country," says Trump. (The Guardian, however, notes that all of the terrorists legally entered the US on tourist or business visas-except one, who used a student visa.) "I'm not blaming George Bush," adds Trump. "But I don't want Jeb Bush to say, 'My brother kept us safe,' because September 11 was one of the worst days in the history of this country." Cue the reactions: "My brother responded to a crisis, and he did it as you would hope a president would do," says Jeb Bush on State of the Union. "He united the country, he organized our country and he kept us safe.... And I don't know why [Trump] keeps bringing this up." "I would probably ask [Trump] what he meant by that. I seriously doubt that he's saying that-that George W. Bush is to blame for [9/11]," says Carson, per ABC News. "I certainly-I certainly don't think so." And Jeb recently fired back at Trump (who's been attacking the Bush brothers on Twitter) with a video that mocks Trump as a foreign-policy lightweight while fanciful clarinet music plays in the background. | 928 | 355 | multi_news | en |
Write a title and summarize: Meta-analysis of genetic association studies increases sample size and the power for mapping complex traits. Existing methods are mostly developed for datasets without missing values, i. e. the summary association statistics are measured for all variants in contributing studies. In practice, genotype imputation is not always effective. This may be the case when targeted genotyping/sequencing assays are used or when the un-typed genetic variant is rare. Therefore, contributed summary statistics often contain missing values. Existing methods for imputing missing summary association statistics and using imputed values in meta-analysis, approximate conditional analysis, or simple strategies such as complete case analysis all have theoretical limitations. Applying these approaches can bias genetic effect estimates and lead to seriously inflated type-I or type-II errors in conditional analysis, which is a critical tool for identifying independently associated variants. To address this challenge and complement imputation methods, we developed a method to combine summary statistics across participating studies and consistently estimate joint effects, even when the contributed summary statistics contain large amounts of missing values. Based on this estimator, we proposed a score statistic called PCBS (partial correlation based score statistic) for conditional analysis of single-variant and gene-level associations. Through extensive analysis of simulated and real data, we showed that the new method produces well-calibrated type-I errors and is substantially more powerful than existing approaches. We applied the proposed approach to one of the largest meta-analyses to date for the cigarettes-per-day phenotype. Using the new method, we identified multiple novel independently associated variants at known loci for tobacco use, which were otherwise missed by alternative methods. Together, the phenotypic variance explained by these variants was 1. 1%, improving that of previously reported associations by 71%. These findings illustrate the extent of locus allelic heterogeneity and can help pinpoint causal variants. Meta-analysis has become a critical tool for genetic association studies in human genetics. Meta-analysis increases sample sizes, empowers association studies, and has led to many exciting discoveries in the past decade [1–5]. Many of these genetic discoveries have informed new biology, provided novel clinical insights [6,7], and led to novel therapeutic drug targets [8,9]. Conditional meta-analysis has been a key component for these studies, which is useful to distinguish novel association signals from shadows of known association signals and to pinpoint causal variants. Existing methods for conditional meta-analysis were proposed based upon the assumptions that summary association statistics from all variant sites are measured and shared. Yet, in practice, the score statistics from contributing studies often contain missing values, possibly due to the use of different genotyping arrays, sequencing capture assays, or quality control filters by each participating cohort. While genotype imputation is an effective approach to fill in missing genotype data for participating cohorts, many scenarios may preclude accurate genotype imputation. For example, a targeted genotyping array/sequencing assay (e. g. exome array) may not provide sufficient genome-wide coverage for imputation. In addition, it is challenging to impute low frequency variants even with the highest quality reference panels. Imputed genotypes of low quality are often filtered out based upon the recommendations from the best practices [10], since these variants are more prone to artefacts and can lead to inflated type I errors. Therefore, missing data in meta-analysis of genetic association studies are unavoidable. Some existing meta-analysis strategies can be highly biased in the presence of missing data. First, a commonly used method for conditional analysis, COJO, can lead to biased results when contributed summary association statistics from participating studies contain missing values [11]. The COJO method approximates the variance-covariance matrix between association statistics with the linkage disequilibrium (LD) information from a reference panel. When the association statistics from contributed studies are missing at some variant sites, the correlation matrix of the meta-analysis statistics can differ greatly from the LD matrix. Consider the simple example of a meta-analysis of two independent studies, where variant 1 is only measured in study 1 and variant 2 is only measured in study 2. The meta-analysis association statistics for the two variants are independent, which cannot be approximated by the LD. COJO only uses meta-analysis results as input. Therefore, it cannot distinguish the scenario where only study 1 measures both variants (and study 2 measures none), and the scenario where study 1 only measures variant 1 and study 2 only measures variant 2. In the presence of missing data, COJO can be highly biased and lead to inflated type I errors. Second, the strategy of imputing missing data from contributed association statistics and using imputed association statistics in meta-analysis can also lead to inflated type I errors in conditional analysis. A simple imputation strategy for marginal (or unconditional) analysis is to replace missing summary statistics with zeros (REPLACE0), which are their expected value under the null hypothesis [2,3]. This method yields valid type I errors for marginal association analysis. Taking this simple approach for conditional analysis, however, is problematic. The genetic variants at conditioned sites are likely to have non-zero effects. Replacing missing summary data with zeros will bias the genetic effect estimates at conditioned variant sites, and can lead to highly inflated type I errors for conditional analysis (see RESULTS). Similarly, the methods that seek to impute missing summary statistics based upon LD (e. g. impG [12]) may introduce substantial biases to the effects of missing variants. Plugging in the imputed Z-score statistics into conditional analysis (impG+meta) can lead to inflated type I errors. Finally, discarding studies with missing summary statistics (DISCARD, or complete case analysis) will give valid type I errors, but at the cost of reduced power. In the statistics literature, synthesis methods have previously been developed to meta-analyze joint effects from different studies, where the participating studies measure different predictors [13,14]. The scenario is similar to the meta-analysis of genetic association studies with missing data. Yet, in genetic association analysis, usually only marginal effects are reported and joint effects have to be approximated from marginal effects. The synthesis methods also lack an implementation for genetic association studies, which greatly limits their impact. To explore the usefulness of synthesis methods, we proposed and implemented an extension of the synthesis methods termed SYN+, which can be applied in genetic association meta-analysis. To overcome these limitations of existing GWAS meta-analysis methods and improve power, we developed an improved conditional meta-analysis method called partial correlation based score statistic (PCBS) that borrows strength across multiple participating studies and consistently estimates the partial variance-covariance matrices between genotypes and phenotypes. We conducted extensive simulations, and showed that our PCBS method has valid type I error and the highest power among all the methods. On the other hand, COJO, impG+meta and REPLACE0 can lead to highly inflated type I errors in the presence of missing data. SYN+, while having valid type I errors, is consistently less powerful than PCBS, especially when the missingness is high or the conditioned variants have larger effects. We also demonstrated the clear advantage of PCBS in the meta-analysis of cigarettes per day phenotype. PCBS identified many more independently associated variants from known loci, compared to alternative approaches. We implemented the proposed methods in the open-source software tools RAREMETAL [15] and R package rareMETALS and made them publically available (https: //genome. sph. umich. edu/wiki/Rare_Variant_Analysis_and_Meta-Analysis). RAREMETAL and rareMETALS use marginal score statistics and exact variance-covariance matrix as input, which is suitable for rare variant association analysis. We also implemented the same method in rareGWAMA (https: //github. com/dajiangliu/rareGWAMA), which conducts meta-analysis using approximate covariance matrix from a reference panel. These methods and tools have been applied and tested in a few large scale meta-analyses. We expect these methods to play an important role in sequence-based genetic studies and lead to important genetic discoveries. We denote the genotype for individual i at variant site j in study k as Gijk, which can take values of 0,1 or 2, representing the number of the minor (or alternative) alleles in the locus. When the genotypes are imputed or generated from low pass sequencing studies, genotype dosage can be used in association analysis. In this case, Gijk will be the expected number of minor (or alternative) allele counts. We denote the non-genotype covariates as Zik, which includes a vector of 1’s to incorporate the intercept in the model. Single variant association can be analyzed in a regression model: Yk = Gjkβj + Zkγk + ek. The score statistic for single variant association takes the form: Ujk=1σ^02∑iGijk (Yik−y^ik) (1) where y^ik=Zikγ^k, γ^k is the covariate effect, and σ^0 is the standard deviation of the phenotype residuals estimated under the null model M0 Yk=Zkγk+ek, ek∼MVN (0, σ^02I) (M0) Without the loss of generality, we assume that the phenotype residuals are standardized in each study as in commonly done in practice. So σ^0 is often equal 1 in practice. We denote the vector of score statistics in a genetic region as Uk = (U1k, …, UJk). The variance-covariance matrix between scores statistics is equal to Vk=1/σ^02[Gk′Gk−GkTZk (ZkTZk) −1ZkTGk] (2) For our illustration of the method, we focus on the analysis of continuous outcomes. Yet, the meta-analysis and conditional meta-analysis methods work for both continuous outcomes and binary outcomes. The meta-analysis score statistics and their covariance matrices are calculated using the Mantel-Haenszel method, i. e. U = ∑k Uk and V = ∑k Vk. The meta-analysis statistics can be used to estimate the joint effects for variants 1, …, J, i. e. β^=V−1U. We denote the score statistics at candidate and conditioned variant sites as U= (UG, UG*), where G and G*represent the genotypes from the candidate and conditioned variants respectively. The variance covariance matrix for U equals to V= (VGVGG*VG*GVG*) The conditional score statistic can be calculated by UG|G*= (UG−VGG*VG*−1UG*) σ^02/σ^c2 (3) where σ^c2 is the residual variance estimated from the conditional analysis model Yk=Gk*βG*+Zkγk+ek, ek∼MVN (0, σ^c2I) (Mc) After conditioning on the genotypes G*, the residual variance equals to σ^c2=σ^02 (1−1NUG*′VG*−1UG*). It is easy to verify that the variance of the conditional score statistics under Mc is equal to VG|G*= (VG−VGG*VG*−1VG*G) σ^02/σ^c2 (4) The single variant and gene-level tests in conditional analysis can be calculated based upon the conditional score statistics UG|G* and the covariance matrix VG|G*. Details are provided in S1 Text. Reviewing formulae (3) and (4), we note that the conditional score statistics and their variances only depend on the partial variance-covariance matrix between the phenotypes and the genotypes after the adjustment of covariates. The key idea underlying our approach is to derive a consistent estimator for the partial covariances in the presence of missing summary statistics and to use it for unbiased conditional analysis. In statistics, to calculate the partial covariance between random variables Gjk and Yk adjusting for variable Zk, we first regress out covariate Zk from both Gjk and Yk, and then calculate the covariance between the residuals. Specifically, ρ^GjkYk|Zk=1Njkσ^02Gjk′ (Yk−Zkγ^) (5) For a given study, it is easy to check that the partial covariances are in fact scaled score statistics, i. e. Therefore, in meta-analysis, we propose to estimate the partial covariance between genotype Gij, phenotype Yi after adjusting the covariate effect Zi using all available summary statistics: ρ^GY|Z, j=∑k∈{k: Mjk=1}Ujk∑k∈{k: Mjk=1}Njk (8) ρ^GG|Z, j1j2=∑k∈{k: Mj1k=Mj2k=1}Vj1j2k∑k∈{k: Mj1k=Mj2k=1}Njk (9) Here Mjk is an indicator variable that takes the value of 1 when the summary statistic at variant site j is measured in study k. For notational convenience, we define the matrices of partial covariance as ρ^GY|Z= (ρ^GY, j) j=1, …, J and ρ^GG|Z= (ρ^GG|Z, j1j2) j1, j2=1, …, J. Under the fixed effect model, we have E (Vk−1Uk) =β for all k. We showed in S1 Text that E (ρ^GG|Z−1ρ^GY|Z) =β. Therefore, the partial covariance matrices can be consistently estimated even in the presence of missing summary statistics. We define partial correlation based score statistics as U˜G|G*=ρ^GY|Z−ρ^GG*|Zρ^G*G*|Z−1ρ^G*Y|Z (10) The covariances for U˜G|G* are equal to V˜G|G*=cov (ρ^GY|Z) +ρ^GG*|Zρ^G*G*|Z−1cov (ρ^G*Y|Z) ρ^G*G*|Z−1ρ^G*G|Z−ρ^GG*|Zρ^G*G*|Z−1cov (ρ^G*Y|Z, ρ^GY|Z) −cov (ρ^GY|Z, ρ^G*Y|Z) ρ^G*G*|Z−1ρ^G*G|Z (11) It is easy to verify that the conditional analysis using the estimator U˜G|G* is equivalent to the standard score statistics when no missing data are present. In the presence of missing data, the partial correlation based statistic U˜G|G* remains consistent. The conditional association analysis can be performed by replacing the standard score statistic with a partial correlation based score statistic. Details for calculating single variant and gene-level conditional association statistics can be found in S1 Text. When the contributed summary association statistics from participating studies contain missing values, a natural strategy is to replace the missing values using imputation. Several imputation methods were previously developed. One method is REPLACE0, which is to replace the missing values by 0. We denote the resulting statistics as U0 and V0. To mathematically describe this method, we define an indicator variable Mjk, which takes value 1 if the summary statistics at site j in study k is measured and 0 if missing. The meta-analysis score statistic is calculated by Uj0=∑k∈{k: Mjk=1}UjkandVj1j20=∑k∈{k: Mj1k=Mj2k=1}Vj1j2k We proved in S1 Text that replacing missing summary association statistics with zero will bias the genetic effect estimate, i. e. E (UG*0) ≠VG*0βG*. As a consequence, under the null hypothesis that the candidate variant is not associated with the phenotype, the expectation of the conditional score statistics is not equal to 0, i. e. E (UG|G*) =VGG*βG*−VGG*0 (VG*0) −1E (UG*0) ≠0. The type I error for conditional analysis can be highly inflated. A more sophisticated set of methods is to impute missing summary statistics based upon LD information. Yet, the genetic effect estimates based upon the imputed Z-score statistics are often biased, unless the following condition holds E[Zimp]=Σimp, tagΣtag−1E[Ztag] where Zimp and Ztag are Z-score statistics at the missing and tagSNP sites, Σimp, tag and Σtag are genotype correlation matrices. A special case for this condition is that both the tagSNP and missing variants have null effects. Similar to REPLACE0, applying impG+meta method can lead to inflated type I errors. We conducted extensive simulations to evaluate the performance of PCBS as well as 5 alternative approaches, including 1) impG+meta; 2) COJO; 3) REPLACE0; 4) DISCARD and 5) SYN+ using simulated data. We simulated genetic data following a coalescent model that we previously used for evaluating rare variant association analysis methods [2]. The model captures an ancient population bottleneck and recent explosive population growth. Model parameters were tuned such that the site frequency spectrum and the fraction of the singletons of the simulated data match that of large scale sequence datasets. For quantitative traits, phenotype data from each cohort were simulated according to the linear model: Yi=β0+∑j=1JGijβj+∑j=1JGij*γj+ϵi where Gij and Gij* denote the candidate and conditioned variant genotypes, and βj and γj are their effects respectively. The model assumes that the genetic variants have additive effects on the phenotype. The genetic effects for candidate variants follow a mixture normal distribution, which accommodates the possibility that a genetic variant can be causal (with probability c) or non-causal (with probability 1 − c): βj∼ (1−c) ×I (0) +c×N (0, τβ2). The genetic effects for the conditioned variants follow: γj∼N (0, τγ2). To evaluate the influence of missing data, we randomly chose a certain fraction (10% 30% or 50%) of the sites from each study and masked them as missing. We then applied the new method PCBS, along with impG+meta, COJO, DISCARD, REPLACE0 and SYN+ to the data. In our evaluations, we used the exact LD with COJO and impG+meta, in order to remove the influence of approximate LD and focus on the impact of missing summary statistics on the power and type I error. We evaluated the type I errors and power for each approach under a variety of scenarios with different genetic effect sizes, fractions of causal variants in the gene region, and the fractions of missing data. To evaluate the effectiveness of methods in real datasets, we applied our methods to a meta-analysis of seven cohorts with a cigarettes-per-day (CPD) phenotype, a key measurement for studying nicotine dependence. Participating studies were the Minnesota Center for Twin and Family Research (MCTFR) [17–19], SardiNIA[20], METabolic Syndrome In Men (METSIM) [21], Genes for Good [22], COPDGene with samples of European ancestry[23], Center for Antisocial Drug Dependence (CADD) [24], and full UK Biobank. Genotypes were imputed using the Haplotype Reference Consortium panel [25] and the Michigan Imputation Server [26] (with the exception of UK Biobank dataset, which was imputed centrally by the UK Biobank team). Summary association statistics from the seven cohorts were generated using RVTESTS [27], and meta-analysis performed using rareMETALS with the PCBS statistics and other alternative approaches. Detailed descriptions of the cohorts are available in S1 Text section 4, including the methods for association analyses and the adjusted covariates. To ensure the validity of our association analysis results, we conducted extensive quality control for the imputed genotype data. We filtered out variant sites with the imputation quality metric R2 <. 7, and sites that showed large differences in allele frequencies from the imputation reference panel. Imputation dosages were used in the association analysis. For each sentinel SNP with genome-wide significance (α = 5×10−8), we defined the locus as the 1 MB window surrounding it. We applied iterative single variant conditional analysis to identify independently associated variants in each locus. We started by conditioning on the most significant variant from marginal association analysis. After each round of the association analysis, if the top variant remained statistically significant, we added the top variant to the set of conditioned variants, and performed an additional round of association testing. We applied the six methods to analyze the data, including the PCBS statistic, SYN+, impG+meta, REPLACE0, DISCARD and COJO. In order to examine if the low frequency variants in aggregate can be explained by the identified independently associated variants, we also performed gene-level association analysis for rare variants with MAF<1%, conditional on the identified independently associated variants. We evaluated the type I errors for the six conditional analysis methods PCBS, SYN+, COJO (with exact LD), impG+meta, REPLACE0, and DISCARD. Scenarios were considered for different combinations of the fractions of missing data, the genetic effects of the variants in the candidate gene, and the genetic effects of the conditioned variants. First, we noted that PCBS, SYN+ and DISCARD are the only three methods that have controlled type I errors across all scenarios, consistent with our theoretical expectation (Table 1). The type I error rate for the other three methods, i. e. impG+meta, REPLACE0 and COJO are inflated in a number of scenarios. The inflation tends to increase with the effect of the conditioned variant (s) and the rate of missingness. In many scenarios, the type I error can be >100X inflated over the significance threshold (α = 5×10−8). For example, when the conditioned variant effect is. 04, and the association statistics from 30% of the variant sites are missing, type I errors for impG+meta, COJO and REPLACE0 are. 015,. 57 and. 74 under the significance threshold of α = 0. 005. When the missing rate is 50%, and the conditioned variant effects is. 08, the type I errors for the three methods become. 25,. 65, and. 60. Second, among the methods with the controlled type I error rates (i. e. SYN+, PCBS and DISCARD), PCBS is consistently the most powerful method (Table 1). The power advantage of PCBS over the other two approaches increases when 1) the conditioned variant (s) have larger effects or 2) the fraction of missing summary association statistics is larger. For example, when candidate variant effect is. 04, the conditioned variant effect is. 08, and the missing rate of score statistics is 30%, the power for PCBS is. 21, which is 75% higher than the power for SYN+ (. 12). When the candidate variant effect is. 08, the conditioned variant effect is. 08, and score statistics from 50% of the variant sites in each participating study are missing, the power for PCBS and SYN+ are respectively. 83 and. 74. Due to the obvious limitations of complete case analysis, the DISCARD method of discarding the studies with missing data can lead to considerable loss of power (Table 1). The power for DISCARD is substantially lower than PCBS and SYN+. In some scenarios where the missingness is high, the power is barely larger than the significance threshold. Interestingly, gene-level association tests are affected by two types of missing data with opposite consequences: Missing values at causal variant sites reduce power but missing values at non-causal variant sites tend to reduce noise and thus improve power (Table 2). When missingness is higher, the power of gene-level tests is lower, but the power loss is small. For instance, when a causal variant in the candidate gene has effects sampled from N (0,0. 22), the conditioned variant has effect. 1, and 30% of the contributed summary statistics in each study have missing values, the power for burden/SKAT/VT tests are 58%/58%/56%, which are only slightly reduced compared to the power of analyzing the complete datasets (60%/61%/60%). On the other hand, the method that discards studies with missing data has much reduced power (0. 011/0. 011/8. 8×10−3). Our method was developed for the fixed effect meta-analysis, where the genetic effects are assumed to be constant across different studies. But since PCBS first aggregates association statistics from across studies and then performs conditional analysis, the impact of genetic effects heterogeneities does not invalidate the test and the type I error remains well controlled. The power is slightly reduced, but the advantages over other methods remain. To confirm this, we performed simulation analysis assuming that the genetic effects across studies are heterogeneous (S1 Table, S2 Table). In our simulations, the genetic effects for a given variant in different studies were simulated from a normal distribution N (μβG*, (μβG*/2) 2), allowing for substantial between-study heterogeneities. The power comparison for different methods remains similar to the scenarios where the genetic effects are the same across studies. We performed a meta-analysis of CPD phenotype in 7 cohorts. The locus CHRNA5-CHRNB4-CHRNA3 was previously identified as associated with CPD [28]. After careful quality control, 42,669,770 variants were meta-analyzed. A majority (32,796,258) of these variants had minor allele frequencies <1%. It is important to note that even with high quality imputation panels, such as the haplotype reference consortium panel [25], there was still considerable missing data in the imputed datasets. A fraction of 76. 1% of the variants were missing from at least one participating study post imputation, due to filtering on the imputation quality (R2>. 7). Compared to common variants, rare variants were considerably more likely to be missing: 95. 3% of the variants with MAF<1% were missing from at least one cohort, compared to the fraction of 20. 1% for the common variants with MAF>1%. The Quantile-Quantile plot for–log10 (p-value) is well calibrated (S1 Fig). The genomic control value is 1. 14 for common variants with MAF>0. 01, and 1. 00 for rare variants with MAF<0. 01. The genomic control value is consistent with that of large scale GWAS for highly polygenic traits [29,30]. The intercept for LD score regression [31] was 1. 01, which shows little influence from potential population structure. The meta-analysis of 7 cohorts identifies 9 loci (S2 Fig), including the well-known CPD associated loci, the nicotine receptor genes CHRNB2, CHRNB3-CHRNA6, CHRNA5-CHRNB4-CHRNA3, the gene CYP2A6 that encodes cytochrome P450 protein, the gene PDE1C that encodes Phosphodiesterase 1C, FAM163B-DBH, YTHDF3 and GRM4. Among these loci, CHRNB2 and FAM163B-DBH are associated with CPD at the genome-wide significance threshold for the first time. While smoking behaviors are known to be heritable, only the CHRNA5-CHRNB4-CHRNA3 and CYP2A6 loci have been consistently implicated in human GWAS to date. The other nicotine receptor gene CHRNB3-CHRNA6 was first identified with genome-wide significance in an isolated population for associations with nicotine dependence and nicotine use [32]. CHRNB2 was implicated in the nicotine dependence trait, but not at genome-wide significance. To our knowledge, there is no report that this gene is associated with CPD at genome-wide significance [33]. In order to understand the allelic architecture of the CPD phenotype and compare different methods on real data, we performed sequential forward selection with the new PCBS method, and identified 5 independently associated variants for the CHRNA5-CHRNB4-CHRNA3 locus and 4 independently associated variants for the CYP2A6 locus at genome-wide significance threshold (with p-values < 5 × 10−8) (Table 3). The other loci do not have additional independently associated variants besides the sentinel variant. As a comparison, we also performed sequential forward selection using the five alternative approaches (S3 Table). Using the SYN+ method, fewer independently associated variants are identified. At the CHRNA5-CHRNB4-CHRNA3 locus, 3 independently associated variants are identified, and also at the CYP2A6 locus, only 3 independently associated variants are identified. DISCARD also identifies fewer number of independently associated SNPs. The results from real data analysis is consistent with our simulation study that PCBS has higher power than alternative approaches. Among the approaches that have inflated type I errors in simulations, impG+meta identifies a lot of SNPs with very significant p-values. Many of these identified SNPs have substantial missingness among the participating cohorts (e. g. N<50,000). Given the inflated type I errors that we observed in simulations, as well as the small available sample sizes for the top variants, the validity of the results using impG+meta is of concern. Most of the top variants identified by COJO and REPLACE0 have low missingness, so there are not many false positive results. Yet, COJO and REPLACE0 identified fewer independently associated SNPs compared to PCBS and SYN+ (Table 3 and S3 Table). Together, the analysis of real data confirmed our simulation experiments. We examined if our independently associated variants explained previously known association signals. To do this, we looked up GWAS catalog [34] using key words “CPD” or “cigarettes per day” and found 11 associated variants in the loci that we identified (S4 Table). We first analyzed these 11 variants conditional on our independently associated variants. All of these variants became insignificant, which indicated that our newly identified independently associated variants can explain previously known association signals. We also performed conditional analysis in the opposite direction to examine if our identified association signal may be explained by the known variants. We found that variants within the CPY2A6 locus remained highly significant and variants within the CHRNA5-CHRNB4-CHRNA3 locus remained marginally significant. Together, our independently associated variants explained 1. 1% of the phenotypic variance, which substantially improves the phenotypic variance (. 64%) explained by the 11 known signals. Finally, in addition to single variant association, we investigated if rare variants within each of the 9 loci were independently associated with the CPD phenotype (S5 Table). 27 genes were analyzed using simple burden, SKAT and VT tests under a MAF threshold of 0. 01. Only one gene (CHRNA5) has gene-level p-values less than 0. 05/27, which is the Bonferroni threshold. None of the genes have exome-wide significant gene-level association p-values. We proposed a simple yet effective meta-analysis method to estimate joint and conditional effects of rare variants in the presence of missing summary statistics from contributing studies. The method leads to the optimal use of shared summary association statistics. It has well controlled type I error and much higher power than alternative approaches even when a large number of contributing studies contain missing summary statistics. Several approaches were previously developed to combine genetic effects across studies when different studies may measure different genetic variants e. g. Verzilli et al [35] and Newcombe et al [36]. These methods have some noticeable limitations. The method by Verzilli et al requires the individual level genotype and phenotype data as input. Also the method focuses on random effects meta-analysis, while our approach focuses on fixed effect meta-analysis. The method by Newcombe et al models the haplotype counts in cases and controls. The method does not allow for the adjustment of covariates, which is a serious limitation. Both methods use MCMC for fitting the model, which may not scale well for contemporary meta-analysis with tens of millions of variants and dozens of studies. It is important to note that our method, PCBS is developed for proper conditional and joint analysis when imputation fails to work. As we showed in our meta-analysis of smoking phenotypes, even with the state-of-the-art imputation methods and high quality reference panels, there are still considerable amount of association statistics filtered out from participating studies. The rate of missingness is much higher for rare variant association statistics than for common variant association statistics. PCBS will be particularly useful for the meta-analysis of sequence data, where the measured variants are predominantly low frequency or rare [37]. Our method is not developed to replace genotype imputation. Genotype imputation fills in missing genotypes with imputed values, and increases effective sample sizes and power. Our method does not increase the effective sample size for tested variants. In practice, imputation method should first be applied in each participating cohort. Our method should be applied at the meta-analysis stage for valid and powerful conditional meta-analysis, especially when contributed summary statistics from participating cohorts contain missing values. Missing data will continue to be a persistent issue in the next generation of large-scale genetic studies. Major biobanks have started to develop their own genotyping arrays and imputation reference panels to incorporate customized content. Combining these newly genotyped studies with existing datasets will result in missing summary statistics. Our method will continue to be useful when analyzing these newly generated datasets. Another major application of the proposed method is in the meta-analysis of sequence data. Given the use of targeted sequencing assays and variability in batch processing and quality control across studies, it would be difficult to impute missing genotype data or missing summary statistics. One of the challenges in sequence-based meta-analysis is to properly represent monomorphic sites, as the polymorphic variant sites are not known a priori. Neither un-called variant sites (e. g. due to insufficient coverage or failed quality control) nor monomorphic sites contribute to the single variant meta-analysis statistic. Yet they should be treated differently in joint and conditional meta-analysis. Summary statistics from monomorphic variants should be replaced by zeros. On the other hand, summary statistics from un-called variants should be treated as missing data, and the conditional association analysis can be performed using our partial correlation based score statistics. While not the focus of this article, the proposed method is also helpful for downstream analyses that make use of the joint effects of multiple variants, e. g. estimating the phenotypic variance explained by variants in LD or fine mapping causal variants (e. g. using methods such as RIVERA [38], FINEMAP [39], CAVIARBF [40]) The validity of these analyses relies critically on the proper estimates of the joint effects, which are usually obtained from single variant association statistics and the LD information from a reference panel. When summary statistics from contributing studies contain missing data, the correlations between resulting marginal meta-analysis association statistics may not be properly approximated by the LD estimated from a reference panel. In this case, PCBS can be used to obtain valid joint effect estimates, which can potentially lead to better calibrated estimates phenotypic variance explained and more accurate fine mapping analysis. Taken together, our partial correlation based score statistic is a simple yet effective method for estimating joint and conditional effects from a meta-analysis. With its efficient implementations in RVTESTS, RAREMETAL and rareGWAMA, this method will have broad application in current array-based meta-analysis, as well as the upcoming imputation-based meta-analysis (e. g. based upon the haplotype reference consortium panel) and sequence-based meta-analysis. Correct inference on the joint and conditional effects using these methods will pave the way for a more accurate characterization and a more complete understanding of the genetic architecture of complex traits. | Title: Proper conditional analysis in the presence of missing data: Application to large scale meta-analysis of tobacco use phenotypes Summary: It is of great interest to estimate the joint effects of multiple variants from large scale meta-analyses, in order to fine-map causal variants and understand the genetic architecture for complex traits. The summary association statistics from participating studies in a meta-analysis often contain missing values at some variant sites, as the imputation methods may not work well and the variants with low imputation quality will be filtered out. Missingness is especially likely when the underlying genetic variant is rare or the participating studies use targeted genotyping array that is not suitable for imputation. Existing methods for conditional meta-analysis do not properly handle missing data, and can incorrectly estimate correlations between score statistics. As a result, they can produce highly inflated type-I errors for conditional analysis, which will result in overestimated phenotypic variance explained and incorrect identification of causal variants. We systematically evaluated this bias and proposed a novel partial correlation based score statistic. The new statistic has valid type-I errors for conditional analysis and much higher power than the existing methods, even when the contributed summary statistics contain a large fraction of missing values. We expect this method to be highly useful in the sequencing age for complex trait genetics. | 8,393 | 287 | lay_plos | en |
Summarize: A groomsman at an Ohio wedding has been arrested for stealing thousands of dollars worth of cash and gift-cards from the newlyweds as they partied with family and friends at their wedding reception. Noah Howard, 29, allegedly stole 55 unopened wedding cards containing a total of $6,800 in cash and gift-cards from a safe at the reception venue - and then went back to celebrate some more with the bride and groom. The theft was discovered after an employee at the Webster Street Market, the reception venue, noticed the cards missing from the safe. Friend indeed: Groomsman Noah Howard allegedly stole $6,800 worth of cash and giftcards from the bride and groom whose wedding he had just taken part in before returning to the reception to party with the newlyweds. According to ABC News, Howard had asked staff if he could change clothes in the office. After the employee discovered the empty safe, the police were called. Before they could arrive, though, the cards were recovered in a Men's Warehouse garment bag that Howard had stashed behind some bushes near the entrance to the venue. Howard told police the money must have been placed in the bag by an employee who tried to steal the cards and then became scared and dumped them in Howard's bag. Joyful occasion: The bride and groom were celebrating their wedding at the Webster Street Market with their nearest and dearest when Howard went to an office to change and emerged with a garment back of cards. AJC reports that surveillance footage shows Howard enter the venue's office to change and then emerge holding the garment bag and swaying as he walked. “It’s just a very extreme thing to do, to take gifts at a wedding,” Mire said, adding he does not know if Howard has financial problems. Howard was booked into the Dayton county jail on suspicion of theft. According to the New York Daily News, Howard has known the groom for 12 years. Police are waiting for the newlyweds to return from their honeymoon before they can complete the investigation | Summary: Noah Howard, 29, is accused of stealing gifts from his close friend and his wife on their wedding day. Howard was a groomsman in their wedding on Saturday. He allegedly stole 55 unopened wedding cards from a safe at the reception venue. After, he went back to the party and celebrated with the bride and groom. The missing cards were noticed by staff who alerted police. The items were found in a tuxedo bag belonging to Howard. He told police that he believes an employee stole them, then became scared and dumped them in Howard's bag. | 456 | 128 | cnn_dailymail | en |
Summarize: By. Harriet Arkell. PUBLISHED:. 08:15 EST, 28 January 2014. |. UPDATED:. 13:16 EST, 28 January 2014. A mother wept in court today as she described the moment her two-year-old girl fell to her death from the fourth floor walkway of an apartment block through a hole allegedly left by a workman. As the trial opened of maintenance worker Robert Warner, 45, jurors were told how Ryaheen Banismuslem wandered off from where she had been playing with her five-year-old brother in a communal garden area at the Wicker Riverside apartments in Sheffield city centre. Her mother, Ola Al Fatle, told the court: 'I didn't let her out of my sight usually. I followed her and she was not there. It was a very short time.' She broke down and sobbed in the witness box for about 30 seconds before still shaking she said: 'I was looking for her but I didn't see. I didn't see anything.' Ryaheen Banimuslem, two, died after falling through a gap in a balcony 'left by workman Robert Warner, 45' The toddler died after falling from the fourth floor balcony of the Wicker Riverside building in Sheffield. 'Then three females said "She was here", and pointed. They were looking down and I saw her down.' Speaking. through an interpreter, Mrs Fatle said she had noticed a gap in the. walkway barrier about two weeks before and and put a bench in front of. it. 'It was clear to all people and was seen to all,' she said. The two-and-a-half-year-old girl was rendered unconscious by the fall and died almost immediately, jurors heard. Mrs. Fatle, who said she rarely used the garden and would not have gone. there if she knew the barrier was missing, said she was planning to. leave the flats because of her safety fears. Court: Ryaheen's mother Olaz Fatle, left, and father Hikmak Banimuslem, right. Mrs Fatle said she rarely used the garden and would not have gone in there if she knew the barrier was missing. Sheffield Crown Court was told that the workman removed the glass panel to use elsewhere in the building. 'It was not comfortable to us as a family,' she said. 'We had nowhere to go, myself and my children.' Earlier, Bryan Cox QC, prosecuting, said Ryaheen had been playing in an outdoor garden area on the fourth floor of the block with her brother, mother and others when she strayed along a walkway and fell through a gap left by Warner. The court heard that Warner, who was contracted to carry out maintenance on the apartments, was supposed to replace a panel in the garden area of the flats, which was regularly used by residents for recreational purposes. Ryaheen, whose father came to England from Iraq to study for a PhD, died after falling 60ft from the balcony. The workman, from Shiregreen, Sheffield, denies one count of manslaughter by gross negligence. But instead of purchasing a new glass panel for the job, prosecutors said he took one from the adjacent walkway, and then invoiced Allsopp Residential Investment Management (ARIM), who oversaw the flat’s maintenance, for the panel he never bought. Mr Cox told the seven female and five male jurors at Sheffield Crown Court: 'This accident was caused by the defendant's negligent conduct. 'The prosecution say he removed the barrier from the walkway, he failed to replace it and did nothing to prevent access to the walkway and created a very dangerous state of affairs and did nothing to warn of any obvious danger.' 'The prosecution say [Warner] removed the barrier from the walkway, he failed to replace it, and did nothing to prevent access to the walkway' - prosecutor Bryan Cox, QC. The prosecutor said Warner, who was employed as a caretaker at the block of flats, had had his hours reduced from five days a week to two. He said a fortnight before the tragedy, Warner had telephoned his manager at ARIM to say two glass panels, one on the ground floor and one on the fourth floor, needed replacing. The works order was given on 14 June, and he invoiced the company for £176 for two panels. The prosecution said it was unclear when the walkway panel was removed, but various witnesses had seen panels missing at different times and the garden panel was broken a'significant' time before 14 June. Warner told police when interviewed that he had checked the flats two days before the girl fell and there were no broken or missing panels. But Mr Cox said the panel was deliberately removed from the walkway, and the only person who had a reason to do so was Warner. Tributes to a young life: Flowers and a class photograph were left at the spot where Ryaheen died. He said: 'He did not intend to injure Ryaheen or cause her death, but the negligence was the cause of her death. It was, say the prosecution, grossly negligent.' The court heard that Warner had more than 20 years' experience working as a builder and maintenance man. He had undertaken courses in health and safety, and his general tasks included maintaining the flats and grounds of the tower block, as well as menial tasks like clearing up rubbish. Warner’s company, RS Enterprises, had been operating for two years and made between £35,000 and £45,000 a year, the court heard. Mr Cox told the jury how Ryaheen was born in Iraq but moved to the UK in 2011 as her father, Hikmat, was studying for a PhD in material physics at Sheffield University. She died almost instantly from her injuries when she fell 60ft on 27 June 2012, the jury was told. Warner, of Shiregreen, Sheffield, denies a single charge of manslaughter by gross negligence. The trial, which is expected to last about three weeks, continues | Summary: Ryaheen Banismuslem, two, fell through gap where a glass panel had been. Robert Warner, 45, had 'taken panel to use elsewhere and not replaced it' Court hears that Warner'moved the panel to avoid buying a new one' Ryaheen fell from the fourth-floor balcony of Sheffield apartment block. Mother Ola Al Fatle tells court how she looked for daughter after she fell. Warner, of Shiregreen, Sheffield, denies manslaughter by gross negligence. | 1,387 | 109 | cnn_dailymail | en |
Summarize: Che il presidente del Brasile Jair Bolsonaro sia contrario ai vaccini anti Covid è cosa nota, così come la sua opposizione a qualsiasi regola di distanziamento sociale: posizioni figlie della sua sottostima della pandemia, ai limiti del negazionismo. Questo nonostante il Brasile sia il terzo Paese al mondo per numero di contagi e conti più di 600mila morti, una cifra impressionante. Tuttavia il presidente di estrema destra non si è fatto vaccinare e ha affermato in diverse occasioni che sarà l'ultimo brasiliano a farlo, se mai lo farà. Niente vaccino, niente passaporto vaccinale e per Bolsonaro domenica sera pure niente partita: "Non può entrare allo stadio, non è vaccinato". È tutto vero, tutto comicamente ma soprattutto tragicamente vero: è successo a San Paolo nei minuti precedenti al fischio d'inizio di Santos-Gremio, sfida del Brasileirao decisiva per la salvezza. Immaginate il nostro Presidente della Repubblica Mattarella che si reca all'Olimpico per assistere a Roma-Juventus e viene rimbalzato all'ingresso dagli addetti ai controlli: "Mi spiace, non può entrare, potrà essere chi vuole lei, ma vada via". Una scena surreale quella avvenuta allo stadio Vila Belmiro, raccontata con sdegno dallo stesso capo di stato brasiliano: "Perché dovrei avere una carta, un passaporto per le vaccinazioni? Volevo solo guardare la partita del Santos e mi è stato detto che dovevo essere vaccinato, perché? Ho più anticorpi io di chi ha ricevuto i vaccini". Bolsonaro si riferisce al fatto di aver contratto il Covid l'anno scorso, tempistica che non lo rende adesso immune dal virus. Bolsonaro era risultato positivo al Covid il 7 giugno 2020, ma la sua posizione scettica – per usare un eufemismo – in merito alla pericolosità del coronavirus non si è spostata di una virgola, sostenendo inoltre che i vaccini sono sperimentali e la loro applicazione non è obbligatoria. Il presidente brasiliano si trovava a San Paolo in vacanza e in precedenza era stato multato per non aver indossato la mascherina in un'area in cui era richiesta. Insomma non il massimo dell'esempio per un Paese dilaniato da un virus che non ha pietà di chi si rifiuta di riconoscerlo. | Summary: Immaginate Mattarella che protesta per entrare all'Olimpico e viene mandato a casa senza tanti complimenti dagli addetti agli ingressi: una scena impossibile, che tuttavia in Brasile è accaduta davvero con protagonista il presidente Bolsonaro. Il capo di Stato brasiliano voleva assistere a Santos-Gremio, ma senza passaporto vaccinale non c'è stato nulla da fare. "Ho più anticorpi io di chi ha ricevuto i vaccini", ha urlato rabbioso. | 492 | 100 | fanpage | it |
Summarize: FIELD OF THE INVENTION This invention relates generally to puzzles and particularly to those utilizing a plurality of interconnected pieces capable of multiple configuration and defining a “solution” configuration to solve the puzzle. BACKGROUND OF THE INVENTION Amusement devices utilizing a plurality of pieces or elements which are capable of variable geometric arrangement are well known in the art. Such devices are typically referred as “puzzles” and most are capable of a variety of orientations and configurations. In such puzzles, the typical solution to the puzzle is found in obtaining a predetermined arrangement or configuration of the puzzle pieces. Often, the external surfaces of the puzzle pieces are variously colored or decorated utilizing a variety of number or letter characters or plural segments of a common picture or artwork. While a great variety of such puzzle amusement devices have been provided by practitioners in the art, such puzzles may be generally divided as either folding puzzles, sliding piece puzzles or those having a plurality of puzzle pieces with interconnecting elements. So-called folding piece puzzles are usually fabricated of one or more planar sheets having pluralities of fold lines and/or edges formed therein. Such puzzles are solved by folding the combination into a predetermined configuration. For example, U.S. Pat. No. 5,735,520 issued to Matos sets forth an FOLD-THROUGH PICTURE PUZZLE which includes a single base sheet, a plurality of superposed attached sheet bases, a single sheet base folded to form a three-dimensional object or plural sheet bases attached to form a three-dimensional object. Each fold-through picture puzzle is continually foldable in a first forward direction and during folding forms assembled images from respective cooperating image portions. U.S. Pat. No. 5,445,380 issued to Polsky sets forth a FOLDING PICTURE PUZZLE having a rectangular multi-picture member which includes a flat base sheet material having a patchwork of partial picture images printed on at least one playing side. The sheet is additionally subdivided into at least sixteen equal and uniform squares by a combination of score lines and cuts. U.S. Pat. No. 4,735,418 issued to Engel sets forth a PUZZLE AMUSEMENT DEVICE having flat strips of equilateral triangles hinged together at their edges. The strips may then be folded at the hinges and end triangles connected together to form a twisted loop having the overall form of a flattened hexagon. Typical sliding puzzles provide some sort of supporting surface often surrounded by a boundary or frame within which one or more puzzle elements are movable between alternative positions. For example, U.S. Pat. No. 4,793,615 issued to Martin sets forth a PUZZLE WITH MOVABLE PIECES having a single plane base on which are mounted movable puzzle pieces. The pieces are restrained in a fixed series of grooves and may be arranged in a desired pattern. The pieces are scrambled in a random arrangement prior to game play which involves moving the pieces to obtain a predetermined arrangement. U.S. Pat. No. 487,318 issued to Clarke sets forth a PUZZLE in which a plurality of pieces are enclosed within a box. Some of the pieces are triangular forming various letter arrangements and combinations to solve the puzzle. U.S. Pat. No. 2,022,319 issued to Meyercord sets forth a MYSTERY PUZZLE having a plurality of pieces which bear segments or portions of a common picture and which are arranged to form the desired picture image solution in combination. Puzzle utilizing interconnected pieces which often comprise polygons and linkages configured to define three-dimensional solutions are also provided in great variety. For example, U.S. Pat. No. 5,108,100 issued to Essebaggers, et al. sets forth a PYRAMID PUZZLE FORMED FROM TETRAHEDRAL AND OCTAEDER PIECES CONNECTED BY A STRAND sets forth a puzzle having a plurality of three-sided pyramids and four small octaeder-shaped bodies all of which are connected to a string forming an endless chain. The solution of the puzzle is obtained by placing the smaller parts of the puzzle in such a manner that a large pyramid is formed which is uniformly colored by the smaller pieces. U.S. Pat. No. 4,483,535 issued to LeCart sets forth a TRIANGLE COMBINATION GAME utilizing a equilateral triangle assembly of hexagonal form subassemblies wherein adjacent subassemblies share two common components and are held together in a manner facilitating rotation of each subassembly around its own center. U.S. Pat. No. 3,981,505 issued to Odier sets forth a PUZZLE WITH IRREGULAR PENTAGONAL PIECES each piece having an identical shape defined by an irregular pentagon. The puzzle pieces may be placed on a planar supporting surface in a side-to-side abutment to fully cover the surface and in a variety of alternate configurations. U.S. Pat. No. 3,201,894 issued to Resch sets forth a GEOMETRICAL DEVICE HAVING ARTICULATED RELATIVELY MOVABLE SECTIONS in which a plurality of three-dimensional objects are interconnected by hinged couplings in a manner facilitating alternative arrangements between the three-dimensional devices. The solution is generally defined as one of a selected type of possible arrangements. U.S. Pat. No. 3,487,578 issued to Sudermann sets forth an ARRAY OF BLOCKS JOINED BY DOUBLE-ACTING HINGE MEANS in which a plurality of equally sized cubes are respectively coupled to adjacent cubes by pairs of opposed crossing filament elements. The opposed pairs of filament elements facilitate the multiple arrangements of the cubes to provide alternate puzzle configurations including a predetermined solution configuration. While the foregoing described prior art devices have generally improved the art and in some instances enjoyed commercial success, there remains a continuing need in the art for evermore interesting, amusing and convenient puzzles and puzzle apparatus. SUMMARY OF THE INVENTION Accordingly, it is a general object of the present invention to provide an improved puzzle. It is a more particular object of the present invention to provide an improved puzzle which is convenient to carry, simple to use and economical to produce but remains challenging in its solution and manipulation. In accordance with the present invention, there is provided a puzzle configurable between a solved configuration and an open configuration, the puzzle comprising: a plurality of puzzle pieces, each puzzle piece having a pair of faceted plates and a detent column joining the pair of plates; and a plurality of linkages, each linkage including a pair of rings joined by a link, each of the rings encircling the detent columns of adjacent puzzle pieces to couple a pair of adjacent puzzle pieces, the detent columns and the rings constructed to cooperatively join the puzzle pieces in a coupling which facilitates rotation of the puzzle pieces individually and rotation of the puzzle pieces about an adjacent puzzle piece. BRIEF DESCRIPTION OF THE DRAWINGS The features of the present invention, which are believed to be novel, are set forth with particularity in the appended claims. The invention, together with further objects and advantages thereof, may best be understood by reference to the following description taken in conjunction with the accompanying drawings, in the several figures of which like reference numerals identify like elements and in which: FIG. 1 sets forth a planar view of the present invention puzzle in a solved configuration; FIG. 2 sets forth a partially sectioned view of the present invention puzzle in an extended configuration; FIG. 3 sets forth a perspective assembly view of a typical piece and interconnecting linkage arrangement; and FIG. 4 sets forth a section view of a pair of puzzle pieces taken along section lines 4 — 4 in FIG. 2. DESCRIPTION OF THE PREFERRED EMBODIMENT FIG. 1 sets forth a planar view of a puzzle constructed in accordance with the present invention and generally referenced by numeral 10. Puzzle 10 is shown in FIG. 1 in its closed or correctly solved configuration. Puzzle 10 includes a plurality of substantially identical puzzle pieces 11, 12, 13, 14, 15, 16 and 17 each having a hexagonal shape and each defining respective surfaces 21, 22, 23, 24, 25, 26 and 27. Puzzle pieces 11 through 17 are substantially identical and, as is set forth below in greater detail, are each formed of a pair of hexagonal plates spaced apart by a detent column such as detent column 105 seen in FIG. 3 for puzzle piece 12. In accordance with the present invention, each puzzle piece is coupled to its adjacent puzzle piece in a straight line arrangement such as seen in FIG. 2 by an interconnecting linkage. Thus, with temporary reference to FIG. 2, it will be noted that puzzle pieces 11 and 12 are coupled by a linkage 31 while puzzle pieces 12 and 13 are coupled by a linkage 32 and puzzle pieces 13 and 14 are coupled by a linkage 33 and so on. The linkage couplings between adjacent puzzle pieces is substantially identical with the exception of puzzle pieces 11 and 17 which, as is better seen in FIG. 2, form opposite ends of the chain of puzzle pieces. In puzzle piece 11, a spacer ring 123 is joined to puzzle piece 11 instead of an additional linkage to an adjacent puzzle piece. Spacer ring 123 is further joined to an extending tab 120 having an aperture 121 formed therein. Aperture 121 may, for example, be joined to a conventional key chain 18 or other suitable carrying device as desired by the user. Similarly, puzzle piece 17, which as is mentioned above is coupled to puzzle piece 16 by a linkage, is an end piece on the chain of puzzle pieces and therefore does not require a second linkage in attachment thereto. Accordingly, a spacer ring 35 having a generally annular shape is received within puzzle piece 17.to properly space the linkage which joins puzzle pieces 16 and 17 (not shown). The remaining puzzle pieces are each mutually joined to an adjacent puzzle piece on each side by a pair of oppositely oriented linkages, each of which is identical to linkages 31 and 32 seen in FIG. 3. With respect to the closed configuration of puzzle 10 shown in FIG. 1, it will be noted that puzzle pieces 11 through 17 define respective surfaces 21 through 27 which in turn support various portions of a combined image. In the example of FIG. 1, a fanciful representation of a custom car is depicted in the combined image. However, it will be apparent to those skilled in the art that other images may be utilized on the surfaces of the puzzle pieces without departing from the spirit and scope of the present invention. It will be further apparent to those skilled in the art that, while not seen in FIG. 1, each of puzzle pieces 11 through 17 is able to support a second combined image on the opposite surfaces of the puzzle pieces in the identical manner to that shown in FIG. 1 for surfaces 21 through 27. As mentioned above and as is set forth below in FIGS. 3 and 4, each of puzzle pieces 11 through 17 is formed of a pair of identical spaced apart plates having hexagonal shapes. Accordingly, plates 41 through 47 of puzzle pieces 11 through 17 are shown interlocking and fitted together in the closed configuration of FIG. 1 which represents a correct “solution” to the puzzle. Once again, while not seen in FIG. 1, it will be understood from the example set forth in FIGS. 3 and 4 of puzzle piece 12 which is understood to be equally representative of puzzle piece 11 and puzzle piece 17 that each of puzzle pieces 11 through 17 includes an identical plate on the opposite side from plates 41 through 47. By way of example and with temporary reference to FIG. 2, puzzle pieces 12 and 13 are shown in partial section having hexagonal plates 52 and 53 which are identical to plates 42 and 43 seen in FIG. 1. In the closed configuration of FIG. 1, puzzle 10 may be carried as a keychain charm or keyfob as desired by the user. Puzzle 10 is played in a problem-solving mode by simply unraveling puzzle 10 from the closed configuration of FIG. 1 to the open configuration of FIG. 2. Thereafter, the user endeavors to pivot puzzle pieces 11 through 17 and restore them to the closed configuration shown in FIG. 1. To add challenge to the solving of puzzle 10, it will be noted that in the manner described below, each pair of hexagonal plates of each puzzle piece is rotatable to a variety of angular positions in addition to the movement about its adjacent puzzle pieces. This adds further interest and challenge to puzzle 10. For example, the arrangement of puzzle pieces in the closed configuration of FIG. 1 could be obtained without the correct rotational position of the puzzle pieces. In such case, the combined image would not be correctly formed and the puzzle even though moved to a closed configuration would not be correctly solved. Thus, the challenge presented to the user is the configuration of the puzzle pieces in the correct manner to form the closed configuration of FIG. 1 and the correct rotational position of each puzzle piece. FIG. 2 sets forth puzzle 10 in an open configuration showing the generally linear arrangement of puzzle pieces 11 through 17. As described above, puzzle pieces 11 through 17 are substantially identical with the exception of the changes to puzzle pieces 11 and 17 in view of their end positions on the puzzle piece chain. Accordingly, puzzle piece 11 includes a hexagonal plate 41 defining a surface 21. Puzzle piece 11 further includes a tab 120 having an aperture 121 formed therein. As mentioned above, tab 120 is secured within puzzle piece 11 by a spacer ring 123 (seen in FIG. 1 ). Puzzle piece 12 includes a hexagonal plate 52, a detent column 105 and a hexagonal plate 42 (seen in FIG. 1 ). Plate 42 supports a post 113 which is received within a bore 112 of detent column 105. The cooperation of post 113 and bore 112 provides the attachment of plates 42 and 52 (plate 42 seen in FIG. 1 ). A linkage 31 includes a link 72 joined to a ring 70 having a substantially annular shape which encircles detent column 105. Ring 70 further supports a plurality of chords 73, 74 and 75, each supporting respective inwardly extending projections 76, 77 and 78. Chords 73, 74 and 75 are preferably formed as integral parts of ring 70 from a somewhat resilient material such as molded plastic or the like. While not seen in FIG. 2, it will be understood that linkage 31 further includes a second ring and plurality of chords identical to ring 70 and chords 73 through 75 as well as projections 76 through 78 which are received upon puzzle piece 11. As mentioned above, puzzle piece 12 supports a detent column 105 having a bore 112 formed therein. Detent column further defines a plurality of detent recesses 106 through 111 (seen in FIG. 3 ). Projections 76 through 78 cooperate with detent recesses 106 through 111 of detent column 105 to detent the relative position between ring 70 of linkage 31 and puzzle piece 12. This detenting action is overcome as puzzle piece 12 and linkage 31 are rotated relative to each other with sufficient force to cause chords 73 through 75 to flex outwardly thereby allowing projections 76 through 78 to be forced from their respective detent recesses at any given rotational relationship between linkage 31 and puzzle piece 12. Thus, a rotation of puzzle piece 12 without movement of adjacent puzzle piece 11 may be attained by overcoming the detent action of puzzle piece 12 and linkage 31. Similarly, the angular or rotational relationship between puzzle pieces 11 and 12 may be changed by moving puzzle piece 11 with respect to puzzle piece 12 with sufficient force to overcome the detent action described above. Puzzle piece 13 is substantially identical to puzzle piece 12 and thus is formed of a hexagonal plate 53 supporting a detent column. 115. A second hexagonal plate 43 (seen in FIG. 1) is secured to plate 53 by a post 104. A linkage 32 includes a link 88 and a pair of rings 86 and 87 (seen in FIG. 3 ). The structure of linkage 32 and its coupling to puzzle pieces 12 and 13 is set forth below in FIGS. 3 and 4 in greater detail. However, suffice it to note here that linkage 32 provides a detented coupling between puzzle pieces 12 and 13 which is identical to the coupling of ring 70 and detent column 105 of puzzle piece 12. A linkage 33 substantially identical to linkages 31 and 32 (seen in FIG. 3) defines a ring 102 and is received upon detent column 115 of puzzle piece 13. Linkage 33 includes a link 103 which is coupled to a ring and chord combination which is not seen in FIG. 2 but which will be understood to be identical to ring 102. Puzzle pieces 14, 15, 16 and 17 are mutually joined by linkages such as linkage 34 in an identical attachment to the attachment between puzzle pieces 12 and 13 to form the remainder of the string of puzzle pieces of puzzle 10. Accordingly, puzzle piece 14 having a hexagonal plate 44 defining a surface 24 is joined to puzzle piece 15 by a linkage 34 correspondingly, puzzle pieces 15, 16 and 17 having respective hexagonal plates 45, 46 and 47 which in turn respective surfaces 25, 26 and 27 are similarly joined by linkages in the manner described above. As mentioned above, puzzle piece 17 differs from puzzle pieces 12 through 16 in accordance with its end position in the string of puzzle pieces by the substitution of a spacer ring 35 for a second linkage which would otherwise further join puzzle piece 17 to the next adjacent puzzle piece were it not in an end position. Thus, it will be understood that puzzle pieces 16 and 17 are joined by a linkage in the same manner as puzzle pieces 12 and 13. Spacer ring 35 maintains the correct position of the joining linkage and makes up for the absence of a second linkage coupled to puzzle piece 17. It will be apparent to those skilled in the art that while the embodiment of the present invention shown is preferred, other embodiments may also be provided within the spirit and scope of the present invention. For example, the present invention is not limited to hexagonal plates. Plates which define different numbers of facets such as pentagons, square and triangular may also be used without departing from the present invention. FIG. 3 sets forth a perspective assembly view of puzzle piece 12 together with the pair of linkages (linkages 31 and 32 ) which are coupled to puzzle piece 12 and which are utilized to further join puzzle piece 12 to adjacent puzzle pieces 11 and 13 (seen in FIG. 2 ). Once again, it will be understood that the structure of linkages 31 and 32 together with puzzle piece 12 is exemplary and illustrative of the identical structures of puzzle pieces 11 and 13 through 17. More specifically, puzzle piece 12 includes a pair of hexagonal plates 42 and 52 defining respective surfaces 22 and 62. Plate 52 supports a detent column 105 having a bore 112 and a plurality of detent recesses 106 through 111 formed therein. Detent recesses 106 through 111 are preferably spaced in an equal angle radial relationship to each other upon detent column 105. In the preferred fabrication of the present invention, plate 52 and detent column 105 are integrally formed of a molded plastic component or the like. Plate 42 defines a downwardly extending cylindrical post 113 which is sized to fit tightly within bore 112. In the preferred fabrication of the present invention, plate 42 is assembled to plate 52 by insertion of post 113 within bore 112 in an alignment which aligns the hexagonal facets of plates 42 with those of plate 52. Attachment of post 113 within bore 112 may utilize a simple tight or force-fit or, alternatively, may use a commercial form of attachment such as chemical adhesive or the like. Linkage 31 includes a link 72 extending between a pair of annular rings 70 and 71. Ring 70 includes a plurality of resilient chords 73, 74 and 75 having respective inwardly extending projections 76, 77 and 78. Correspondingly, ring 71 supports a plurality of resilient chords 80, 81 and 82 supporting respective projections 83, 84 and 85. Linkage 32 is identical to linkage 31 and includes a link 88 supporting a pair of rings 86 and 87. Ring 86 includes a plurality of resilient chords 90, 91 and 92 supporting respective projections 93, 94 and 95. Similarly, ring 87 includes a plurality of resilient chords 96, 97 and 98 supporting inwardly extending projections 99, 100 and 101. Puzzle piece 12 is assembled by initially placing ring 86 of linkage 32 upon detent column 104 and thereafter placing ring 70 of linkage 31 upon detent column 105 and thereafter assembling plate 42 to plate 52 by insertion of post 113 into bore 112. Once again, it will be understood that adhesive attachment or the like may be used to secure post 113 within bore 112. It will be further understood in the preferred embodiment of the present invention, plate 42 is aligned with plate 52. FIG. 4 sets forth a partial section view of puzzle 10 taken along section lines 4 — 4 in FIG. 2. FIG. 4 shows puzzle pieces 12 and 13 commonly joined by linkage 32. FIG. 4 also shows in part the further attachment of puzzle piece 13 to puzzle piece 14 (seen in FIG. 2) by linkage 33. Also shown in FIG. 4 is a portion of linkage 31 which joins puzzle piece 12 to puzzle piece 11. More specifically, puzzle piece 12 includes a pair of plates 42 and 52 having respective outer surfaces 22 and 62 joined by detent column 105. Similarly, puzzle piece 13 includes a pair of plates 43 and 53 having respective outer surfaces 23 and 63 joined by detent column 115. Also, partially shown in FIG. 4 is puzzle piece 11 having plates 41 and 51 defining respective outer surfaces 21 and 61. A linkage 32 defines rings 86 and 87 received upon detent columns 105 and 115 in the manner described above which provides the detented attachment coupling between puzzle pieces 12 and 13. Linkage 33 includes ring 102 received upon detent column 115 together with link 103. Linkage 31 includes a ring 70 received upon detent column 105 together with a link 72 joined to a ring 71. Thus, it can be seen from FIG. 4 that puzzle pieces 12 and 13 are commonly joined by linkage 32 and that puzzle piece 12 is further joined by linkage 31 to puzzle piece 11 while puzzle piece 13 is further joined to puzzle piece 14 (seen in FIG. 2) by a linkage 33. Once again it will be understood that the couplings and structure of puzzle pieces 12 through 16 are identical to the coupling shown between puzzle pieces 12 and 13 in FIG. 4. It will be further understood that puzzle piece 17 (seen in FIG. 2) is identically coupled to puzzle piece 16 in the manner shown in FIG. 4 with the difference being found in the replacement of ring 102 of linkage 33 with spacer ring 35 (seen in FIG. 2 ). Similarly, it will be understood that the coupling of puzzle piece 11 to puzzle piece 12 utilizes linkage 31 together with spacer ring 123. What has been shown is a simple puzzle structure which nonetheless provides an interesting and amusing puzzle to be solved by the user. The puzzle provides substantial interest and challenge and in the closed or solved configuration may be further employed as a typical keyfob. The components of the inventive puzzle are readily fabricated of molded plastic components for economy and ease of manufacture. While particular embodiments of the invention have been shown and described, it will be obvious to those skilled in the art that changes and modifications may be made without departing from the invention in its broader aspects. Therefore, the aim in the appended claims is to cover all such changes and modifications as fall within the true spirit and scope of the invention. | Summary: A plurality of hexagonal puzzle pieces are formed of a pair of spaced apart hexagonal plates having a detent column extending between their respective inner surfaces. A plurality of linkages each having ring portions are received upon the detent columns of each puzzle piece to join the puzzle pieces in an elongated string. The angular positions between adjacent puzzle pieces as well as the rotational position of each puzzle piece within the string of puzzle pieces may be changed or manipulated in order to configure the puzzle pieces in a closed or solved puzzle configuration. A detent mechanism is operative between each puzzle piece and its adjacent puzzle piece to detent the relative positions thereof at selected angular relationships. | 6,129 | 142 | big_patent | en |
Summarize: In the midst of a weak attempt at rebranding herself as "healthy," Food Network personality Paula Deen is launching her own line of butter at Walmart. After revealing her diabetes diagnosis a year and a half ago, Deen had been trying to move away from her image as a lying liar, pharma shill, shameless recipe thief, and private jet aficionado. In that light, butter may seem like an odd choice for her, but never fear, Deen's new butters are actually, really, truly being marketed as healthy. They're "finishing" butters that "let cooks bring a wonderful fresh butter taste to various dishes while just adding butter to the end of the cooking process," and they come in five flavors: A Look at Paula Deen's Butters [Photos: Neil Chopra/Eater] · Southern Grilling Butter ("Garlic goes with everything.") · The Lemon Dill Butter ("Perfect for a tangy and creamy grilled chicken y'all.") · The European Style Butter ("Y'all know that I love my travels to Europe and I call my European Style Butter 'Bread's Best Friend.'") · Sweet Citrus Zest Butter ("My Sweet Citrus Zest butter is hard to practice in moderation...it feels so fresh and clean on your palette [sic]." · Garden Herb Butter ("The herbs just explode in my mouth.") She also is launching a line of tortilla chips and sugar free chocolates, available at Walmarts in the Southeast. Video: Paula Deen on Today Yesterday, Deen made a couple appearances on television in which she used butter at the end of a recipe, as opposed to her old schtick of all-butter-all-the-time. It's healthier that way, or something? This is a woman who has a recipe for deep fried butter balls that takes two and a half hours to prepare. What happened to Deen not wanting to "associate her name with butter anymore?" About a year ago, on HGTV's Celebrities at Home, Deen said, "When you hear the name Paula Deen...I want you to think of the word 'hope.'" Hope you like compound butters. Anyway, she never mentioned her Walmart line in her appearances. Instead she appears to be laying last minute ground work to make it seem like finishing butter is just a thing that she does. Instead of promoting a product on television (which producers would likely not allow), she's selling the concept. Stopping by Today, she tells Matt Lauer, "I know you're going to find this heard to believe, but I've been trying to cook with less butter...I'm trying not to use butter during the cooking process. I wait til the end, and then I put the finishing butter on it." At no point does she mention she has a line of finishing butters at Walmart. "By the way, that still counts," Lauer astutely points out. Deen retorts, "But it only counts once instead of like six times." Video: Paula Deen on The Chew Next it was on to The Chew, where Deen made ribs with Mario Batali. They were finished with butter, obviously: "Y'all this is what I've been trying to do lately. I've been trying not to cook with butter like through the whole thing, but I just take the finishing butter and I finish up." The succotash was also finished with butter, which Batali points out got its fat from non-butter sources: "Instead of using that pesky butter, we use three pounds of bacon fat and then we finish it with a little bit of this finishing butter." Again, on neither The Chew nor Today did Deen mention she was selling finishing butters. Both The Chew and Today were a large part of her diabetes announcement tour. Deen announced her diabetes diagnosis and pharma sponsorship on Today on January 17, 2012. The next day, doing damage control, Deen went on The Chew and said she would donate an undisclosed percentage of her pharma profits. Video: Paula Deen on Paula Deen Foods Deen also released a truly bizarre video via Twitter in which she talks about why she is launching her food line. It has to do with death, and memories: "So with the food company, it's given me an opportunity to encourage families to come back to the kitchen together and build those memories, because you never know when you're going to have to count on those to get you through the day-to-day struggles." Buy Paula Deen's butter, because some day your family will die? Here's the full transcript and the video: Because of different things that have happened to me in my life, y'all, it really brought me to this stage in my life with a few passions. You know, I lost my folks early on when my brother and I were both very young, so I really, really, really count on my memories to fill in the gaps for me and to kind of complete my family. So with the food company, it's given me an opportunity to encourage families to come back to the kitchen together and build those memories, because you never know when you're going to have to count on those to get you through the day-to-day struggles. It's my hope to bring affordable, every day luxuries to these young mothers and fathers out there raising families, and to older couples that don't do a whole bunch of cooking any more. And you know one of the best parts of this whole Paula Deen Foods is a percentage of the profits are going to my new foundation called the Bag Lady Foundation. So it makes me very, very happy to know that I'm going to have a small part in paying it forward to others in their struggles. And I thank y'all so much for considering my products. I will try my best to never, ever let you down. Butter profits are going to charity, y'all. Here's the press release: PAULA DEEN FOODS MAKES A SPECIAL OCCASION FOR EVERYDAY MEALS America's Favorite Chef Brings the Magic of Her Traditional Recipes & Lighter Fare to Leading Retailers Across America Savannah, GA (June 5, 2013) – America's favorite cook, restaurateur, author and entrepreneur Paula Deen has launched her own line of food products at retail; Paula Deen Foods (www.pauladeenfoods.com), based on her traditional Southern recipes and lighter fare as well. Her initial product line will include all-natural tortilla chips made with fresh vegetables and a line of compound "finishing" butters that let cooks bring a wonderful fresh butter taste to various dishes while just adding butter to the end of the cooking process. Leading retailers Walgreens and Walmart will be featuring the all-natural chips and finishing butters, respectively. As well, Deen brings a very special benefit to this new venture, as a portion of the proceeds from the sales of her products will be donated to the organization that she has helped found, The Bag Lady Foundation (www.thebagladyfoundation.org}, which has a mission of serving women and families in need. "It is such an honor to be able to not only let the everyday cook bring special fresh meals, quality ingredients and great-tasting snacks to their tables to share with their friends and families, but it is an overwhelming joy to able to give back to communities across the country through The Bag Lady Foundation," says Paula Deen Foods co-founder Paula Deen. "It is my dream that in the years to come, I'll be inspiring the next great cooks to enjoy the best ingredients and products available today with their families, friends and those they love." It was just a little over two decades ago that Deen herself was challenged with supporting her family, as a single mom with two young boys. She was fighting agoraphobia and the only way she could make money was to cook in her own home and send her young boys door-to-door selling bag lunches and her first business "The Bag Lady" was born. Just a few decades later, Deen has launched this new venture in partnership with Buffalo, New York-based Nanco Group, whose principal Steven Nanula was involved with the successful Northeastern retailers Tops Neighborhood Market & Wilson Farms. "We're honored to bring Paula's great products and recipes to American families to provide fresh meal solutions with the best ingredients that make every day eating a special occasion," says Paula Deen Foods CEO, Steven Nanula. In 2013, in addition to her all-natural chips and finishing butters, Deen will be bringing a line of sugar-free and premium chocolates to the marketplace with these confections having been developed from her special private blend of chocolate. Late last year Deen launched her chocolates on QVC and the sales of her sugar-free line were tremendously successful. · Paula Deen Foods [Official Site] · Paula Deen and Sons Grill Up Quick and Easy Steak [Today] · Paula Deen's Camp Menu [ABC] · All Paula Deen Coverage on Eater [-E-] Whether you’re an apple or a pear-- or even a banana--Paula Deen wants to make sure you look your best while cooking in the kitchen or just out and about. The southern dining diva launched a new line of clothing with digital shopping channel Evine this week. And while it make seem like an unusual next step, the move from the pantry to the runway has been a long time coming for the former Food Network star. “I’ve been trying to do this for years,” Deen told People magazine. “ And one of my most asked questions out there — whether it’s in person, through the mail, or on Facebook — is ‘Paula, where did you get that top? Where did you get that outfit?’ So I wanted to do something like this because I understand the body that’s not perfect. You see all these things made for bananas. Well, I’m an apple. Most women are an apple or a pear. There are not many bananas.” Deen’s line consists of tops, dresses and pants in fits that flatter all sizes of women, according to Evine’s website. Designs include ombre knits, stretchy, breathable maxis and kimono sleeve tunics. Deen says that once women hit 30, they often don’t have “perfect bodies” anymore so she wanted a line to “accentuate your good points and try to camouflage your bad ones.” In typical Deen fashion—pun intended—the queen of comfort cuisine had some specifics when it came to choosing patterns for her pieces. According to People, she wanted to make sure tops provided enough coverage to avoid any wardrobe malfunctions and that they hit below the hips. “I don’t know how to say this to you in a nice way, but it has to cover my front privates,” said Deen. “I am aware of ‘catfish belly’ arms. Have you ever seen a catfish? You know how they have a smooth floppy belly on them? Well, that’s what we refer to as ‘catfish belly’ arms.” If you, too, need to cover up your catfish belly arms, you can find several items from Deen’s collection online. Prices range from $35 for tops to $60 for longer dresses. This is the chef's second collaboration with the digital shopping platform. Last year, Deen launched a line of hams, cake mixes and chocolate butter sticks. Deen will appear on Evine throughout the month to promote her new clothes. MINNEAPOLIS, Feb. 16, 2016 (GLOBE NEWSWIRE) -- EVINE Live (NASDAQ:EVLV), a digital commerce company (evine.com), today announced that it will expand its partnership with Paula Deen – celebrity chef, restaurateur, author and two-time Emmy Award Winner – by premiering Paula Deen’s Home on March 13 and 14. First joining the digital retailer in 2015 with a line of kitchen and pantry products, Paula Deen has quickly become an EVINE Live fan-favorite. Paula Deen’s Home builds on the success of her kitchen and pantry line with a sophisticated offering of home décor. A photo accompanying this announcement is available at http://www.globenewswire.com/NewsRoom/AttachmentNg/e855893e-ca4b-46fe-bc0d-a50af9c4a82f “Paula Deen is known best as the ‘Queen of Southern Cuisine,’ but few know that she’s also an accomplished painter, fashionista and a connoisseur of home décor,” said Penny Burnett, Chief Merchandising Officer of EVINE Live. “Our product development team partnered closely with Paula when designing this collection, taking inspirations from her art, style and Savannah, Georgia home. Paula Deen’s Home truly embodies Paula’s personal approach to design and we’re excited to introduce her millions of fans to this new line.” “Working with EVINE Live and connecting directly with my fans has been a wonderful experience,” said Paula Deen. “I approach style the same way I approach cooking. You need the right ingredients to make it all work. A pinch of color, a dash of texture and the perfect mix of comfort and sophistication. I’m looking forward to sharing all of my passions with EVINE Live’s customers – my love of art, style and design.” Paula Deen’s Home will launch on March 13 and 14 with an assortment of sophisticated bedding. During the premiere, Paula will present a number of colorful designs inspired by her art and her home, including patterned and embroidered coverlet sets, comforter sets, embroidered bed sheets and cotton blankets. The chic, comfortable style of Paula Deen’s Home is designed to add an element of charm and comfort to any bedroom. Viewers are invited to shop, share and smile and tune in to EVINE Live via cable and satellite, mobile apps and live streaming online at www.evine.com. EVINE Live airs on DIRECTV Channels 73 and 316, DISH Network channels 134 and 228 and the nation's top cable providers. Find EVINE Live in your area: bit.ly/1CNa450. For more information on Paula Deen’s EVINE Live collections, visit www.evine.com/PaulaDeen. For more information on EVINE Live, visit www.evine.com. About EVINE Live Inc. EVINE Live Inc. (NASDAQ:EVLV) is a digital commerce company that offers a compelling mix of proprietary and name brands directly to consumers in an engaging and informative shopping experience via television, online and on mobile. EVINE Live reaches approximately 88 million cable and satellite television homes 24 hours a day with entertaining content that engages its community of customers in a comprehensive digital shopping experience. About Paula Deen Paula Deen has sold over 11 million copies of her 14 cookbooks. Deen’s first live tour, “Paula Deen Live!” commenced in June 2014, and featured cooking demonstrations, games, and stories with Paula and her fans. In September 2014, Paula Deen Ventures launched the “Paula Deen Network," a subscriber-based interactive digital experience that broadcasts a variety of exclusive content featuring Paula anytime, anywhere. She showcases her signature Southern dishes and simple, healthy meals in a fun, unscripted format on a daily basis with all-new recipes, episodes and cooking tools. The Lady & Sons, the Savannah, Georgia restaurant Paula founded with her sons, Jamie and Bobby Deen, remains one of the country’s most popular regional restaurants. On March 11, 2015, Paula launched her first free mobile game, Paula Deen’s Recipe Quest. In April 2015, she opened her new restaurant, Paula Deen’s Family Kitchen, in Pigeon Forge, TN. In May 2015, Paula Deen Ventures launched Paula’s podcast, Get Cooking with Paula Deen, and radio show, What’s Cooking with Paula Deen. On September 8, 2015, Paula released her new cookbook, Paula Deen Cuts The Fat: 250 Favorite Recipes All Lightened Up. Also in the fall of 2015, Paula competed in the twenty-first season of ABC’s hit show “Dancing with the Stars,” and successfully made it halfway through the competition. Deen’s robust social media following includes Facebook (4.5 million likes), Twitter (1.43 million followers) and Pinterest (273,300 followers). Paula Deen Ventures is headquartered in Savannah, Georgia, with offices in New York. The photo is also available at Newscom, www.newscom.com, and via AP PhotoExpress. Contacts Media: Dawn Zaremba EVINE Live Inc. press@evine.com (952) 943-6043 For Paula Deen: Jaret Keller Key Group Worldwide jkeller@keygroup.tv (212)988-7701 Investors: Jason Iannazzo EVINE Live Inc. jiannazzo@evine.com (952) 943-6126 Paula Deen, southern cookbook queen, former Dancing With the Stars contenstant and butter’s biggest fan, has taken a lot of bold steps in her career. But her most recent step is one away from the pantry and towards the walk-in closet, with the debut of her brand new clothing line, Paula Deen’s Closet, which she launched with Evine this week. Surprised? Don’t be — it’s been a long time coming. Courtesy of Evine “I’ve been trying to do this for years,” Deen told PeopleStyle today. “It was a little hard for me to break out of the kitchen because that’s how people think of me. But if you notice, I never cooked naked. I was always wearing clothes! And one of my most asked questions out there — whether it’s in person, through the mail, or on Facebook — is ‘Paula, where did you get that top? Where did you get that outfit?’ So I wanted to do something like this because I understand the body that’s not perfect. You see all these things made for bananas. Well, I’m an apple. Most women are an apple or a pear. There are not many bananas.” Courtesy of Evine That’s why she made her line of tops, dresses, and pants full of flattering fits, from ombré knits to stretch maxis to kimono sleeve tunics. “When you get up over 30, most of us don’t have those perfect bodies anymore,” she said. “You have to accentuate your good points and try to camouflage your bad ones. I want to cover all body types. I am not trying to please just one person. I’m trying to please a multitude of people with this line. And I think people trust me. They know that I will not lie to them … To the best of my knowledge, I will tell the truth.” RELATED: Paula Deen’s New Outlook on Weight Loss Therefore, she created the line with a few priorities in mind. First, she took great concern with the length of the tops and making them long enough to hit below the hips. “I don’t know how to say this to you in a nice way, but it has to cover my front privates,” she said. “I want my shirttail to cover my front privates and hopefully my back, too. I am aware of ‘catfish belly’ arms. Have you ever seen a catfish? You know how they have a smooth floppy belly on them? Well, that’s what we refer to as ‘catfish belly’ arms.” Cleavage is also a focus, particularly not showing any. “When I’m doing V-necks, I want that placement perfect so you can lean over and you don’t have wardrobe malfunctions,” she said. “I don’t like when you show everything.” The price is also important to her; everything is under $60. “Just like when I opened my very first restaurant, I told my sons, ‘We want to serve the very best meal that we’re capable of preparing at a fair price’,” she said. “So everything I do must have value. People must feel like they are being treated fairly. I’m not interested in trying to gouge anybody.” RELATED: Paula Deen Says ‘Challenges Are What Help Build Character’ Finally, she wanted all her items to be comfortable. “I like to think that I’m comfortable to be around, and that definitely transfers into the clothing line,” she said. “And at the stage I’m in, comfort is important. That’s definitely the way I feel about my shoes. I think so many young girls kill their feet with their shoes.” RELATED VIDEO: What Paula Deen Cooked for Her Dancing With the Stars Co-Stars That’s not the only thing that she finds perplexing about the PYTs of today. “I’m quite puzzled when I see a lot of things,” she said. “There are a lot of funky styles out. I had to get used to the jeans with rips in them. But now I’m cool with it. I get it now. But that took me a while to understand why you’d want to wear things with holes on them.” However, she still hasn’t gotten on board with the super-revealing styles on red carpets today. “Definitely [young people are] showing a lot more than when I was their age,” she said. “Do you think the Kardashians are somewhat responsible for that? I don’t know. I mean I don’t know them. But the role models are there that these girls have to follow, they want to be trendy too.” Paula Deen will return to the Evine studios today to promote her line, and you can see the entire line on evine.com. —Sharon Clott Kanter Paula Deen is back—and she’s working hard to reclaim her title as the queen of Southern cuisine. Last fall, Deen premiered her subscription-based, digital platform, the Paula Deen Network and just last month released a mobile game Paula Deen’s Recipe Quest. She's even returned to TV after being dropped from the Food Network in 2013, debuting her own show on Evine Live, a broadcast shopping network, where she's offering her own line of branded food products. Up against big names like QVC and the Home Shopping Network (HSN), Deen now has a slimmer, (35 lbs to be exact) healthier image, but hasn't given up the Southern comforts. “Deen’s camp approached us with a new idea and it took us less than five minutes to jump on board,” Mark Bozek, the CEO of Evine, told FoxNews.com. “She has a large loyal fan base that is definitely excited to see back.” Among Deen’s new food line are two versions of her popular Ooey Gooey Cake—in both butter and chocolate flavors, two types of smoked sausages, holiday hams and savory glazes. She also has a line of chocolate candies which come in a sugar free version, a nod to Deen’s struggle with Type 2 diabetes. More on this... New goodies from Paula Deen We tried out her original Ooey Gooey Cake mixes in the butter flavor and were happy with how easy it was to replicate an authentic Deen recipe. It was really sweet, but that’s to be expected. But the finished cake came out looking just like the box. One of the wackiest items in Deen’s new food collection is chocolate shaped like the ingredient Deen is most known for —butter. It comes in three flavors, milk, dark and white chocolate. Initially, we weren’t sure if these were chocolate flavored butters or a Deen original recipe. But a quick clarification from the Evine team set the record straight. They're not considered a baker’s chocolate, though they can be added to recipes, used as shavings or melted to drizzle on top of treats. But really, they’re just a funny shaped Hershey bar. So how do they taste? Sweet. Very sweet. The dark chocolate, though pleasantly smooth, lacked the cocoa punch we’re used to with deeper chocolates. The milk and white were nicely creamy but lack any complexity. Our biggest gripe with the product is how difficult it is to cut or bite. At about an inch around, biting into a cube of chocolate is no easy feat. We had to melt ours to get a little chunk to nibble. So as a snack this might be too much work but works as a novelty item—the package even says “Hey Y’all” with a smiling picture of Deen. And for Evine, which also sells a line of seafood caught by the fisherman on “Deadliest Catch,” having items that get people talking is a good thing. Deen’s appeal reaches way beyond what she bring to the table. “Part of what she does is just have fun—there’s no question about it,” Bozek says. “We’re in the business of entertaining customers and having that kind of personality—that puts a smile on your face—just goes with what we do.” Preparation Cream the butter, cream cheese, salt and pepper together with an electric mixer until smooth. Using a very small ice cream scoop, or melon baller, form 1-inch balls of butter mixture and arrange them on a parchment or waxed paper lined sheet pan. Freeze until solid. Coat the frozen balls in flour, egg and then bread crumbs and freeze again until solid. When ready to fry, preheat oil in a deep-fryer to 350°. Fry balls for 10 to 15 seconds until just light golden. Drain on paper towels before serving. Yields about 30 balls. Paula Deen is adding fashion to her repertoire. The Southern cooking guru already has a kitchen, pantry and home line at digital commerce company Evine Live. The latest addition — Paula Deen’s Closet — effectively makes her a lifestyle name at Evine. According to Deen, “This is always something I wanted to do. One of the main questions on my Web site is ‘Where did you get that top?’” Deen said she has known for a long time that there is a demand from fans for what she is wearing. “If I have something that all my friends out there want, I want to be able to bring it to them for a reasonable price. Evine [said they are] happy to launch my clothing line. I think I share my body with a lot of women out there who are not a size 4. We want nice-looking, comfortable clothes. They look like they’re dry cleaned but are washable,” Deen said. Evine’s design team heads to Deen’s home periodically to check out her closet for inspiration for the line. Deen’s wardrobe is extensive, and the closet in her Savannah, Ga., home is about “18 feet by 20 feet,” with most of the space taken by the height of the room, she said. The room utilizes a “moving dry-cleaning rack” to take advantage of the room’s height, Deen noted. The first showing of the capsule line on Evine is slated for Monday afternoon, with another showing for two hours Tuesday night. Deen said the plan is to add new items to the line each month. So far jackets, bottoms and tops comprise the line. Price points range from $34 to $55 for tops, up to $60 for dresses and outerwear, such as duster coats and sweater coats. With the kitchen, home and fashion covered, what’s next for Deen? She said she’d like to pursue jewelry for her next product line. For the last two years, Evine has been making a bigger push to include celebrity-based lines that are exclusives on its site. On the fashion side, more recent additions include Vanessa Williams, Nancy O’Dell and Karen Fairchild. About the Collection Travel to Savannah, Georgia and discover the styles and flavors of Paula Deen, featuring kitchen and pantry items, clothing and home décor from the famed “Queen of Southern Cuisine.” Known best for her southern charm and mouthwatering recipes, Paula Deen is also an accomplished painter, fashionista and home style connoisseur. Approaching her décor and clothing with the same creativity and passion as her cooking, Paula’s tasteful styles are created with just the right ingredients: a pinch of color, a dash of texture and the perfect mix of comfort and sophistication. About the Guest Culinary icon and queen of southern cooking, Paula Deen is a self-made success who learned her culinary secrets from her grandmother. Deen's first business, The Bag Lady, started her career and led to the opening of her first restaurant, The Lady and Sons, which she opened with her two boys, Bobby and Jamie, five years later. In addition to her success on TV and with her restaurant, Deen is the author of 14 cookbooks that have sold more than 11,000,000 copies. In 2014, Deen launched the Paula Deen Network, an interactive digital cooking network that combines cooking, lifestyle and game shows with great recipes, meal-planning tools and more. | Summary: Paula Deen, who's had her own line of butter, has sold chocolate butter sticks, and is known for recipes like Paula's Fried Butter Balls, has now launched a clothing line that will help you hide the fact that you've consumed 35 sticks of butter in a day. Paula Deen's Closet, described on its website as "Southern comfortable," will cover you in all the right places, the celebrity chef promises. "I want my shirttail to cover my front privates and hopefully my back, too. I am aware of 'catfish belly' arms," Deen explains to People. "Have you ever seen a catfish? You know how they have a smooth floppy belly on them? Well, that's what we refer to as 'catfish belly' arms." "I understand the body that's not perfect. You see all these things made for bananas. Well, I'm an apple. Most women are an apple or a pear. There are not many bananas," she adds. She also promises all the pieces will be comfortable, cost $60 or less-and "look like they're dry cleaned but are washable," she tells Women's Wear Daily. She launched the collection with Evine, a digital shopping channel, Fox News notes. She already had a line of home products with Evine, and in a press release announcing that in February, an Evine exec assures us that Deen is a true "fashionista" (as well as, randomly, an "accomplished painter.") | 6,531 | 344 | multi_news | en |
Summarize: A homeless man who received a surprise meal and hotel stay after spending a $100 donation on food for his friends has insisted that no part of the experience was a hoax - despite the fact one witness is claiming the whole thing was a set-up. The man - named only as Thomas - had been filmed living on the streets of California, before being taken to a sushi restaurant, given a hotel room with hot tub and treated to a new suit and haircut. But the resulting video posted online by'serial prankster' Josh Paler Lin was met with varying degrees of skepticism, with a number of viewers dismissing it as a hoax. One witness, in particular, has insisted that there was no way that Thomas did not know he was being filmed as in the original video he arrived at the liquor store in the same car as Paler Lin. SCROLL DOWN FOR VIDEOS. Smartened up: In Josh Paler Lin's newsest video, homeless Thomas, who became an internet hit after spending a $100 donation on food for his friends, is given a new suit and a makeover. Nursing student Taugan Tan Kadalim, 26, told Vocativ that he was outside the liquor store on the day in question and he saw Paler Lin - who is recognized from his previous videos - in the driver's seat. In the passenger seat, he claims, was Thomas. Kadalim told the site: 'While I think the guy is homeless, it is clear that from what I saw every part of that scene was staged.' However, Thomas has now spoken to Fox LA, insisting that to him it was 'all a surprise' and he had never met Josh before he was handed the $100. He told the news channel: 'I appreciate it, most definitely. It's all a surprise to me. I thought this was just a project video, I had no idea this was going where it went. Thomas added: 'Normally people give you a dollar, maybe five as a big tip, but a hundred, wow that's a week's worth. Crazy.' Paler Lin had initially set out to 'expose homeless people' in Los Angeles by giving them cash in the expectation that they will spend it on alcohol. However, Thomas proved to be an exception. Instead, he spent the money on food to share with his friends, prompting Paler Lin to return and surprise Thomas with a Christmas meal, makeover and a night in a hotel. Gesture... with an agenda? Skeptics have picked holes in a viral video showing a homeless man spending a $100 donation on food for his beggar friends, deeming it a 'fake' creation that was focused on making money. The feel-good videos, that have gained more than 26million views, are part of a campaign to raise more than $120,000, via an Indiegogo page for Thomas to get his life back on track. But critics pointed to a number of apparent flaws in the footage, as well as the hefty profit Paler Lin has reportedly made from its success - up to a huge $52,000. YouTube users have been skeptical about Paler Lin's motivation, suggesting it is a'set-up' so he can gain traffic and profit. One YouTube user, with the handle ClemtheRanter, said: 'A large number of YouTube prank videos are staged,' he said. 'I was involved in a bloody eyeball scare prank by Josh Paler Lin. My part of the video was staged... me getting scared by Josh. Using chopsticks: Lin captioned this photo of Thomas: 'Taking Thomas to eat Shabu Shabu in this Xmas eve' Twist: The short film sees'serial prankster' Josh Paler Lin give $100 to a beggar named Thomas in Los Angeles, California - only for him to walk into a Liquor Mart (left) and buy food for his struggling friends (right) 'I asked him if he could give me a shout-out and put my name in the description below so I can get some traffic to my YouTube channel, but it never happened. 'So now i'm addressing this issue due to a number of YouTube pranksters getting exposed for making stage pranks.' Other users have claimed that homeless feel-good films are a sure-fire way of boosting traffic - and consequently, money, via advertisements - on the social networking site. 'They go to a homeless person and make a feel-good video because they know that's going to get views,' said one YouTube user, referring to the site's personalities, such as Paler Lin. Skepticism: Now, viewers have questioned the video's authenticity, citing a number of 'flaws' in the footage. Above, Lin is pictured in a Facebook photo. He is well-known for producing 'prank' videos for YouTube. Indeed, in November 2013, Paler Lin gave another homeless man $100 and told an actor playing a beggar to approach him to ask for money for food. Again, the real beggar handed over some cash. The user, with the handle '24/7 rants', claimed the start of Paler Lin's recent homeless video - which sees the director hand Thomas $100, before secretly following him with a camera - immediately suggests it may be a hoax. Speaking in his own video, he said Paler Lin could not possibly have guaranteed the man was going to pick up his things and spend the money immediately, instead of remaining by the roadside. 'How did he know he was going to spend the money immediately? He had no idea,' he said. He also picked holes in the claim that Thomas did not notice the camera - nor Paler Lin (who has a distinctive haircut) - following him throughout the duration of his trip to a nearby Liquor Mart. After shopping at the Mart, Thomas was filmed emerging with bags filled with food, before going straight to a park, where he handed out the goods to a number of homeless people. Wit his new look and wardrobe, Thomas was treated to a buffet meal at the end of the day by Paler Lin. However, the user questioned the beggar's choice of a Liquor Mart a 34-minute walk away for his food shop - instead of a nearer, cheaper supermarket - saying: 'It seems like an odd coincidence'. 'Why would this guy, of all places, go to a liquor store to buy chips and pie or whatever when you think that there are tons of other places to go to... that are convenient, probably cheaper. However, Paler Lin argued that Thomas chose the liqour store because it was closest to his begging spot, saying: 'When people see you getting all this traffic they want to bring you down.' When questioned by DailyMail.com, he did not explain why Thomas chose to drag several bags of food 34 minutes to the park, instead of shopping at least two stores that were closer to the park. But he strongly denied staging any of his films or having met Thomas before the $100 exchange. 'I had little expectation, maybe he would buy alcohol maybe he wouldn't,' he said of the video. 'I wanted to see what a homeless person would spend it on. No one has ever done that before.' Giving: The camera following Thomas as he hands food to a family in the park he was initially filmed in. However, the YouTube user claimed the number of hits received by Paler Lin's previous YouTube videos - between 600,000 to 800,000, according to socialblade.com, a site that tracks YouTube channel statistics - could have prompted him to construct what he knew would be a viral hit. Indeed, Paler Lin - who earns money based on how many users view his videos, most of which are 'pranks' - not only scooped $52,000 from the film, but also gained 13,000 new subscribers. After the video went viral, Paler Lin said on YouTube: 'I wasn't expecting to get this kind of footage. To be honest, I thought this video would be more an exposing homeless people video at first.' He added: 'I'm so glad that I could witness and capture such a beautiful moment. This has to be one of the most amazing experience so far on this channel. In the footage, Thomas told Paler Lin he had quit his job to look after his parents, but after both of them died, he couldn't afford to pay for their condo and found himself on the street. 'The more I talk to him, the more I sense how genuine he is…. I gave him my number and told him to call me when he needs help. Never judge a book by its cover. One love!' Paler Lin said. Another video: In November 2013, Lin (pictured, left) gave another homeless man (right) $100 and told an actor playing a beggar to approach him to ask for money for food. The video gained far fewer views than Thomas's | Summary: YouTube star Josh Paler Lin filmed himself giving beggar Thomas, $100. Video showed Thomas spending cash on food for his homeless friends. In second clip, Palen Lin treats the homeless man to a number of luxuries. Skeptics have picked holes in the footage, deeming it a 'fake' creation. Thomas insists he had never met Paler Lin before, events were not set up. Paler Lin told DailyMail.com the events were natural and not pre-planned. | 2,048 | 112 | cnn_dailymail | en |
Summarize: BACKGROUND OF THE INVENTION This invention relates to apparatus for forming pads of material from a more compressed form of the same material, and in particular to apparatus for forming the pads in the shape and contour required, including varying the thickness across the width and length of the pads, without the need for later cutting and trimming and the resultant waste. The conventional method for forming fibrous pads begins by feeding a felted web of compressed material such as wood pulp into a hammermill, where it is defibrated to a light, fluffy material called "fluff". This fluff is then drawn through the screen of the hammermill and onto a screen belt by a source of vacuum below the belt. There is thus formed on the belt a fluff blanket of relatively uniform thickness of about one inch, more or less, across the entire width of the belt, which can be up to 24 inches or more. This blanket of fluff is then conveyed continuously downstream to machines which process it into the particular end products being manufactured, whether they be feminine hygiene pads, disposable diapers, adult incontinence products or other products employing fluff pads. Conventionally, the processing includes cutting the blanket into the proper shape for the particular product being manufactured, and then wrapping and packaging it for later use. Cutting the blanket into the proper shape, however, can result in a substantial waste of fluff. For instance, to form a product such as a feminine hygiene pad, the blanket must be cut into oval shapes. Generally this is done by use of a rotary die cutter. This creates a "dog-bone" shaped waste between the ends of the respective ovals. If on the other hand the product is a disposable diaper or adult incontinence product, no waste is created by the cut between the pads, since the edges are straight. The sides, however, are usually cut in the shape of an hourglass to allow space for the legs of the wearer without causing bunching. The cuts required to allow space for the legs again cause waste of fluff. Moreover, the mere fact that the fluff blanket must be cut at all is a substantial disadvantage. As to the rotary die cutter mentioned above, the tolerances for cutting fluff are extremely close, and so the maintenance costs can be very high. Additionally, any fluff that is cut using a rotary die cutter is left with a very hard edge, since the fluff is very tightly compressed at the edge by such a cutter at the time of cutting. Because such a hard edge would be objectionable for the leg cutouts of disposable diapers and adult incontinence products, due to the skin irritation such a hard edge would cause, water-jet cutting has come to be the conventional method of forming the "hourglass" cuts required, because it results in a fluffy, soft cut edge. Of course water-jet cutting has its own disadvantage, that is, extremely high maintenance costs. These high costs arise from the fact that the water must be forced at extremely high pressure through a fine nozzle:, causing the nozzle to experience high wear rates almost regardless of the material from which the nozzle is made. Further, for the water to be reused in a continuous cutting operation, it must be carefully filtered to avoid clogging the nozzle. A further disadvantage of the above-described system lies in the screen belt on which the fluff blanket is formed. This screen belt is tautly stretched over the rollers on which it runs. The fact that the screen itself runs on the rollers and is pulled across a vacuum box, which has a perforated top cover, produces friction which causes wear to the screen. As the screen wears, fine bits of metal wire may wear off and may become entangled in the fluff blanket. This situation may give the unsatisfactory result that diapers and feminine hygiene pads may have bits of metal in them, which would cause great irritation or worse. The entire process of forming a uniform fluff blanket and then cutting fluff pads therefrom has at least one other significant disadvantage: the uniformity of the blanket itself. Thus for instance the absorbent padding of a disposable diaper is the same thickness at the waist portion, which seldom gets wet, as it is between the legs where much moisture must be absorbed, because the thickness of the blanket from which it was cut was uniform throughout its length and width. Finally, referring to the waste of fluff alluded to above, the raw material used to make this fluff, such as wood pulp, is expensive. Costly machines have been designed and sold simply to recycle, reprocess and reuse this fluff cut out of the shapes described above to avoid discarding this waste and the cost associated therewith. A patent to Furbeck, U.S. Pat. No. 3,717,905 discloses an apparatus for forming fibrous pads, although that apparatus employs a seal roll as a required component. Two other patents, one to Savich, U.S. Pat. No. 3,939,240 and one to Kolbach, U.S. Pat. No. 3,973,291, disclose methods for forming fibrous pads. Kolbach's method includes a screened belt, which slides over multiple vacuum boxes, with all of the wear problems attendant thereto. Savich's method includes forming larger pads and passing them through smaller openings as a means of forming and compressing the pads. Applicant has discovered that the pads, once formed, do not hold their shape well if later compressed or forced through smaller openings. This invention relates to solutions to the problems described herein. SUMMARY OF THE INVENTION This invention includes a pulp defiberizer, such as a conventional hammermill into which is fed a felted web of compressed, fibrous material, such as wood pulp. The defiberizer grinds the material into a fine, fibrous material called fluff. This fluff is then drawn through a screen in the defiberizer, and deposited onto an interchangeable screen form, called an "insert", by a source of vacuum below the insert. This insert is in the shape of the pad desired, whether that shape be oval for feminine hygiene pads, hourglass for diapers, or any other desired shape. After passing under scarfing rolls, which remove excess fluff, the insert moves to an area where it is shielded from vacuum, and the pad formed therein is removed and the wrapping and packaging of the pad accomplished. Ideally, a group of inserts are assembled in the shape of a wheel so that the process of thus forming the pads can be continuous. In addition to forming the proper outline of the pad, the insert can be constructed so as to form a pad which is thicker in some areas and thinner in others, so that more material is deposited where needed and less where it is not needed. It is therefore one object of this invention to provide an apparatus for forming fibrous pads in the shape in which they will be used, without the need for later cutting. Another object of this invention is to provide an apparatus for forming a fibrous pad having varied thickness over its surface. Yet another object of the invention is to provide an apparatus which vacuum forms fibrous pads by use of interchangeable inserts. Still another object of this invention is to provide an apparatus employing inserts, for forming fibrous pads, which inserts have a foraminous upper surface, such as a screen, supproted by a foraminous floor which moves along with the screen to minimize wear to the screen. Other objects and advantages of this invention will become apparent hereinafter. DESCRIPTION OF THE DRAWING FIG. 1 is an elevation of an apparatus embodying the invention. FIG. 2 is a sectional view of FIG. 1 taken along line 2--2. FIG. 3 is an enlarged view of a portion of FIG. 1. FIG. 4 is a further enlarged view of a portion of FIG. 1. FIG. 5 is an isometric view of a wheel formed by the inserts and backing plate, partially cut away. FIG. 6 is a top view of an insert which forms part of one apparatus embodying the invention. FIG. 7 is a sectional view of FIG. 6 taken along line 7--7. FIG. 8 is a sectional view of FIG. 6 taken along line 8--8. FIG. 9 is a top view of an insert which forms part of another apparatus embodying the invention. FIG. 10 is a sectional view of FIG. 9 taken along line 10--10. FIGS. 11-15 show a few of the shapes of pads which can be formed by the use of the invention. FIG. 16 is an enlarged view of the takeoff roll and scarfing rolls also shown in FIG. 1. FIG. 17 is a sectional view of FIG. 15, taken along line 17--17. FIG. 18 is a side view of a pad shown in top view in FIG. 15. DESCRIPTION OF THE PREFERRED EMBODIMENTS Very generally, a web of compressed fibrous material 100, shown in FIG. 1, is fed into hammermill 170, fiberized, and the fibers entrained in an air stream caused by vacuum source 235. As shown in FIG. 2, the air stream draws these fibers down onto depressions on the upper surfaces of inserts 240, which are partially foraminous, in the shape and contour of the pad desired. Referring again to FIG. 1, inserts 240 pass under scarfing rolls 245, where the excess fibers are taken off and recycled via stack 255. inserts 240 and the pads therein then pass under takeoff roll 265, where the pads are taken from the inserts and dropped onto a conveyor 275 for wrapping and packaging. Referring now in more detail to FIG. 1, the embodiment of the invention there shown accepts compressed fibrous material, such as wood pulp, from rolls 110 and 120 on stands 130 and 140, which employ braking devices 150 and 160. While the Figure shows two rolls, meaning that the material is being fed in two thicknesses, the material can be fed in any reasonable number of thicknesses, and can even be fed via sheets as opposed to rolls. The material 100 is fed into a hammermill 170 between feed rollers 180. Hammermill 170 is generally conventional. Any hammermill generally available which can handle compressed fibrous material, such as the Meteor model from Williams Patent Crusher and Conveyor Company, of St. Louis, would be suitable. In this instance, hammermill 170 rests on crossbeams 185 which in turn are supported by posts 190. The hammers 200 and grinding plate 210 of hammermill 170 work together conventionally to break down the material 100 into its individual fibers, or as nearly thereto as possible, which fibers are then collectively called "fluff". This fluff is drawn through the screen 220 of the hammermill 170 and into a funnel 230 by a vacuum source 235, via a path which can be best explained by reference to FIG. 2. Referring now to that Figure, the path of the vacuum is from hammermill 170 through its screen 220 and through inserts 240. The vacuum then is turned toward the back of the apparatus (that is, away from the viewer in FIG. 1) and passes through openings 250a in the back of backing plate 250 and openings in the frame 260, behind which the vacuum source 235 (FIG. 1) has its inlet 236 (FIG. 2). Referring again to the path of the fluff, the vacuum draws the fluff down from the hammermill to be deposited on the foraminous upper surface of insert 240, as can be seen best in FIG. 2, as the vacuum passes through this upper surface and exits the back of the machine as explained above. As shown best in FIGS. 3 and 5, each insert 240 is arcuately shaped and, together with a number of identical inserts 240, form a drum 270. This drum 270 may preferably be located below the hammermill, and its axis may preferably be centered below and parallel to the axis of the hammermill. As shown in FIG. 5, each insert 240 is removably secured to the edge of backing plate 250 by any suitable means. One preferable means, shown herein, is to thread pegs 280 into plate 250. Inserts 240 can then be formed with a matching semi-cylindrical groove 240a in each end. The spacing of pegs 280 about plate 250 is such that each insert 240 fits tightly between its respective pegs. The length of each peg should be nearly but no greater than the width of the inserts. After each insert is slid between its respective pegs, a bolt 290 and washer 300 are tightened into the outer end of each peg to hold each insert tightly in position. Alternatively the inserts could be simply bolted to plate 250 or affixed in any other secure but removable manner. Referring again to FIG. 2, plate 250 is fixedly secured to an axle 310, which passes through frame 260 via bearings 320. Axle 310 is then connected to a suitable prime mover 330, such as an electric motor, via a suitable speed reducing arrangement 340, such as reducing gears, or appropriately sized pulleys and belts. Journaled to axle 310 in front of plate 250, by bearing 350, is a front sealing plate 360 having a rough "S" shape. Front plate 360 includes a semicircular outer portion 360a, a hub portion 360b and a semicircular recessed portion 360c. Hub 360b contains the bearing 350 and is secured to the outer and recessed portions or formed integrally therewith. Outer portion 360a is aligned just outside of the front surface of inserts 240, while recessed portion 360c is aligned just outside of the front surface of plate 250. Finally, connecting portion 360d, shown by broken lines in FIGS. 3 and 4, runs between the outer and recessed portions of the front plate 360 from the hub 360b to the outer edges of the plate. Front plate 360 is prevented from rotating by any suitable means, such as a bracket 370, which is attached to plate 360 and to frame 260. Finally, a shroud 380, attached to frame 260, surrounds the entire upper portion of the drum 270. The front surface 380a of the shroud 380 is approximately planarly aligned with upper front plate 360a while the rear surface 380b is approximately planarly aligned with plate 250. The purpose of shroud 380 is to provide an area through which the fluff can be dispersed before it is drawn into the inserts, and to close the vacuum path around the inserts. Inserts 240, once filled with fluff, pass under scarfing rolls 245, which can be bristle brush rolls as shown in FIG. 3 or any other type of roll having an abrasive surface. As shown in more detail in FIG. 16, the fluff 247 will normally pile above the upper surfaces of inserts 240. The purpose of scarfing rolls 245, then, is to remove this extra fluff 247 from each pad so that the top of the pad is even with the top surface of insert 240. The scarfing rolls accomplish this function by spinning at high speed in the same direction as drum 270, such that the surface of the roll rubs against the upper surface of inserts 240 and removes the extra fluff therefrom. To facilitate the recirculation of the loose fluff fibers through stack 255 and hammermill 170 once they are taken off the inserts 240 by scarfing rolls 245, air vanes 382 and 384 and air inlets 386 and 388 are provided inside shroud 380, and are shown best in FIG. 3. In effect, vane 382 separates the area where the fluff pads are formed from the area where the inserts are scarfed and the excess fluff recirculated. As shown by the arrows in FIG. 3, a substantial amount of air enters the apparatus at inlet 388, while a lesser amount enters at inlet 386. Most of the air which composes the airstream carrying the fluff down to inserts 240 comes from inlet 388 originally, and gets to hammermill 170 via stack 255. The purpose of air inlet 386 and vane 384 are to provide a small amount of air flow downward and to the right in FIG. 3 for the purpose of picking up the excess fluff as it is taken off inserts 240 by rolls 245, and carrying the fluff into the air flow caused by inlet 388 for recirculation. Both vanes 382 and 384 reach the entire width of shroud 380, from front surface 380 to rear surface 380b. As drum 270 continues rotating, it next meets with takeoff roll 265. This takeoff roll has a foraminous surface similar to the surface of the inserts, except that the screeing is at the surface rather than recessed. Takeoff roll 265 rotates about its axle 266 at a rate such that the surfaces of it and drum 270 are synchronized. Journaled to axle 266 is a cover plate 267 which is extremely similar to cover plate 360 which covers drum 270, in that cover plate 267 also has an outer portion 267a, a hub 267b, a semi-circular recessed portion 267c and a connecting portion 267d, shown in FIG. 16 in broken lines, which runs between the outer and recessed portions from the hub 267b to the wheel 265. The parts of cover plate 267 can be formed integrally or secured together by bolts or other means. Similar to cover plate 360, cover plate 267 has vacuum behind the outer portion 267a, which draws air through the part of the takeoff wheel 265 which is behind it. Then, as inserts 240 pass connecting portion 360d of cover plate 360 and move into an area where there is no vacuum behind them, pad 268 is taken out of the insert by takeoff wheel 265 because of the vacuum behind it. The pad 268 then adheres to wheel 265 until it passes to the area where no vacuum is applied, that is, past right connecting portion 267d, and, since the pad is no longer held to takeoff wheel 265, the pad drops onto conveyor 275 to be conveyed to the downstream processing machinery for wrapping and packaging. Of course, at the start-up or shut-down of an apparatus such as this, certain of the pads may be improperly formed and so a means for culling these pads, that is, keeping them from going onto conveyor 275, is desirable. Hence, cover plate 267 is not fixed in any one position the way cover plate 360 is. Rather, the position of this cover plate is controlled by a cylinder 269, one end of which is pivotably attached to a bracket 267e on cover plate 267 and the other end pivotally attached to a support bracket 271 which is bolted to frame 260. Cylinder 269 may be actuated by air, hydraulics, or any other suitable means. In the position shown in FIG. 16, as described above, the pads 268 drop onto conveyor 275 to be further processed. When cylinder 269 moves to its extended or "cull" position (not shown), however, cover plate 267 rotates clockwise up to 90 degrees. Since the near end of conveyor 275 does not reach as far as the center of wheel 265, the area behind outer portion 267a of cover plate 267 would then not be over conveyor 275, and so the culled pads would drop to the floor below rather than onto conveyor 275. When the pads are again properly formed, cylinder 269 is returned to the position shown in FIG. 16 and pads 268 drop onto conveyor 275 for processing. As stated above, drum 270 continues to rotate. As it does so, inserts 240 next move away from takeoff roll 265 and toward a cleaning box 372, which is supported in bracket 370. Box 372 applies reverse vacuum to the upper surfaces of inserts 240, which are at that point the bottom surfaces because inserts 240 are upside down at the bottom of drum 270. Box 372 is connected to vacuum source 235 by hosing 374. Hence, any bits of fluff not taken out of inserts 240 by takeoff wheel 265 with pad 268 are cleaned out by vacuum box 372, making the apparatus essentially self-cleaning and selfmaintaining. Since the entire system is under vacuum it must be sealed, at least to an extent, against the entry of air between the various parts discussed above. The seal need not be complete and total, and in fact it may be advantageous and desirable to have a very small amount of air enter at the various joints between parts. Wholesale entry of air at these points would tend to degrade the level of vacuum, however. Hence, felt or other equivalent seals are provided at various points to keep most of the non-evacuated air out, and are held in place by suitable means. Accordingly, a large seal 400 connects funnel 230 to shroud 380, shown best in FIGS. 2 and 3, and is supported by brackets 405, shown in FIG. 2, which are in turn connected to crossbeams 185. These brackets 405 also provide some support for funnel 230 and shroud 380. Smaller seals 410 close the gaps between shroud 380 and inserts 240 as shown in FIG. 2. Also, seals 420 close the gaps between inserts 240 and front plate 360, while seal 430 reduces air leakage between backing plate 250 and frame 260. One of the many possible embodiments of inserts 240 can be seen by reference to FIGS. 6, 7 and 8. The pad to be manufactured using the embodiment shown is of an oval shape although it is formed as a double oval to be folded into a single oval during later processing, possibly after the insertion of super-absorbing material. A top view of insert 240 is shown in FIG. 6. The necessary foraminous surface is here formed by a screen 440. Screen 440 is partially cut away to show holes 240b beneath, through which the air under vacuum passes. FIG. 7 is a lengthwise sectional view of the insert 240 shown in FIG. 6. As can be seen in FIG. 7, the contour of insert 240 is an arc of a circle such that a number of inserts 240 can be assembled to form the circumference of a complete drum. Accordingly, holes 240b and sides 240c are formed along lines which radiate from the center of the circle. Screen 440 is set a certain distance 460 below the top surface 240d of insert 240. This distance 460 below surface 240d at which screen 440 is set determines the thickness of the pad being formed. Insert 240 can be molded in a single mold wherein the distance 460 is determined at the time the mold is made. The preferable method, however, is to mold a separate cover 240e and base 240f, place the screen 440 between the two, and permanently adhere them together, such as with epoxy or other suitable adhesive to form insert 240. The upper surface of base 240f then provides a floor for supporting screen 440 without rubbing or wearing the screen itself. A cross-sectional view of FIG. 6 is shown in FIG. 8. As can be there observed, the way the insert 240 is formed and the way the screen 440 is supported by areas of the base 240f between holes 240b allows the distance between the screen 440 and top surface 240d, and hence the thickness of the pad formed thereby, to vary as necessary. Thus for instance one part of the pad can have thickness 460 while another part can have thickness 460a. Any necessity for cutting or shaping the pad thereafter has been eliminated. Of course, the variance in the thickness of the pad referred to above is determined and set at the time the insert 240 is formed. Once the screen 440 is secured within insert 240, no variance in the distance 460, or in the thickness of the pad formed by insert 240 is possible or even desirable. Another embodiment of an insert to be applied in the instant invention is shown in FIGS. 9 and 10. FIG. 9 is a top view of an insert 480. Insert 480 is used to directly form an oval pad, with no further processing other than wrapping and packaging. As was the case for insert 240, the foraminous surface is here formed by a screen 500. FIG. 10 is a sectional view of insert 480. As can there be seen, screen 500 is again supported within insert 480. Similar to insert 240, insert 480 again is formed having rounded grooves 480a for removably attaching insert 480 to plate 250 by sliding it onto pegs 280. Again, the holes 480b and exterior edges 480c are radial, while the upper and lower edges 480d form an arc of a circle, such that a number of inserts 480 can be assembled to form a complete drum. And again insert 480 can be molded as one part with screen 500 in place, or can be molded as a cover 480e and base 480f and permanently affixed together after 480f provides a floor for supporting screen 500 without rubbing or wearing the screen. Notice that in FIG. 10 the distance 510 from the top surface 480d of insert 480 to screen 500 varies smoothly from a relatively small distance at the ends to a relatively larger distance near the center. This insert 480, then, would form an oval-shaped pad which is thicker near the center than at the edges, without the necessity of any later cutting or folding. This same principle is applied advantageously without regard to the actual shape or contour of the pad to be made. FIG. 11 shows a group of pads 520 formed using insert 480. FIG. 12 similarly shows a group of pads, 530 formed using insert 240. FIG. 13 shows a continuous stream 540 of pad material which could be formed using a group of appropriate inserts. The purpose of an embodiment which forms such a continuous stream of pad material would be to adapt the apparatus disclosed herein to conventional downstream equipment which would cut as well as process and package the individual pads. Shown in FIGS. 14 and 15 are the hourglass-shaped pads 550 and 560, used in making baby diapers and adult incontinence products. Pads 550, shown in FIG. 14, are each formed at a predetermined spacing whereas pads 560, shown in FIG. 15, are formed close together and later separated before wrapping and packaging. Pads 550 and 560 were formed applying the same principle, employing an insert having a screen which was further below the top of the insert in the middle than at the ends. Hence in FIG. 17, which is a longitudinal section of a pad shown in FIG. 15, it can be seen that there is more fluff material in the center of the pad than at the edges. Similarly, in FIG. 18, which is an end view of a pad shown in FIG. 15, it can be clearly seen that the pad 560 is substantially thicker at the center than at the sides. While the apparatus hereinbefore described is effectively adapted to fulfull the aforesaid objects, it is to be understood that the invention is not intended to be limited to the particular preferred embodiments of pad-forming apparatus herein set forth. Rather, the invention is to be taken as including various equivalents without departing from the scope of the appended claims. | Summary: An apparatus for defiberizing a web of compressed material into fluff and then forming the fluff into pads, and a method of use of the apparatus. The apparatus includes a hammermill or other machine for performing the actual defiberizing. The fluff is then drawn by vacuum which is contained within a shroud, into insert molds which form fluff pads in any shape and contour desired. The insert molds are assembled into a drum which turns continuously such that the vacuum is applied to only certain of the molds. As each mold leaves the shroud, any excess fluff which may overfill the mold is removed and recycled. The pad is then removed from the mold and passed downstream for further processing. The apparatus includes an arrangement for vacuuming out any remaining bits of fiber after the pad is removed. Also included is an apparatus for culling the pads at the discretion of the operator for start-up and shut-down as well as other purposes. The excess fluff which overfills the molds can be removed by scarfing rolls and additionally by air streams which are drawn by the vacuum and directed at the excess fluff as it is removed by the scarfing rolls. The molds themselves are changeable and can be easily replaced if a different shape or size of pad is required, by merely removing the current insert molds and installing ones having the desired shape and contour. | 6,947 | 297 | big_patent | en |
Summarize: The relatives of a baby boy who was brutally killed by his parents today condemned their short nine-year jail term as a ‘kick in the face’. Levi-Blu Cassin, of Smith's Wood, Birmingham, died as a result of horrific abdominal injuries ‘consistent with being hit by a car or falling from a three-storey building’. But, following a five-week trial, his parents Danielle Cassin, 27, and Mark Piper, 31, were both cleared of murder and manslaughter after a jury was unable to pinpoint which one had dealt the fatal blows. Scroll down for video. Defendants: Following a five-week trial, parents Danielle Cassin (left), 27, and Mark Piper (right), 31, were both cleared of murder and manslaughter after a jury was unable to pinpoint which one had dealt the fatal blows. They were sentenced to just nine years in prison at Birmingham Crown Court yesterday for causing, or allowing, the 22-month-old’s death in February last year. Levi-Blu’s grandmother, Angela Cassin, told today how the boy’s family are ‘devastated and disgusted’ by the sentence and condemned social services for failing to prevent his death. She said: ‘No amount of justice will bring him back – but nine years is like a kick in the face. We’ve just lost so much. 'We don’t know the truth – and we’re never going to know the truth. We’re forever having different scenarios running through our heads.’ During the trial Cassin – a known drug addict - from Chelmsley Wood in Solihull, West Midlands, and Piper, of no fixed address, blamed each other for the death of their son. A year after his death, the pair remained together and refused to tell police of the drugs and violence that plagued their son’s life. Death: Levi-Blu Cassin (left), died as a result of injuries 'consistent with being hit by a car or falling from a three-storey building'. Also pictured (right) is a memorial garden set up in his grandmother's back garden. To this day neither has revealed how he suffered his ‘major internal injuries’ - comparable to those seen after car crashes or falls from two or three storeys. Throughout the trial the court heard how Cassin would take her son to crack dens where she would take Class A drugs for hours at a time. And Piper was painted as a violent bully who regularly beat her. The drugs and violence at their flat prompted neighbours and friends to call Solihull Social Services and police. But Levi was never taken away from his parents. A Serious Case Review - to be published in the spring - is now investigating whether police and social services failed to protect the boy. In a statement issued yesterday, following the verdict, Levi-Blu’s family said: ‘We refuse to let Levi-Blu be another sad statistic that we only remember when another poor innocent child is murdered. ‘This was a tragedy that could have been prevented if the right steps had been taken by the various agencies responsible for the care and protection of our beautiful angel.’ On camera: A CCTV video of Cassin spotted trading goods at a second-hand store in Chelmsley Wood, West Midlands, while Levi was alone with Piper in their flat. The footage was shown to jurors in court. Family: (From left) Levi-Blu's uncle Tyler Cassin, aunt Kirsty Cassin, grandmother Angela Cassin and uncle Kurtis Cassin. Mrs Cassin, 47, who lives around the corner from where her daughter and grandson lived, said yesterday: ‘Danielle could have said anything to me and I would have helped her. ‘She could have just given me Levi, and said she was having problems - I would have taken him. I always believe life is about choices and this is what she chose to do. Levi had no choices.’ She added: ‘I just want the world to love him and know how much he was loved.’ Birmingham Crown Court heard how Cassin phoned for an ambulance after her son stopped breathing at around 4.30am on the morning of his death. Paramedics arrived at the couple’s flat to find him lying on the floor between the cot and his mother’s bed. Prosecutor Timothy Raggatt QC said the lifeless child’s eyes were open but his lips had turned blue and his heart was not beating. He was rushed to hospital on blue lights but was pronounced dead at around 5.30am on February 20 last year. Memorial garden: The drugs and violence at the boy's flat prompted neighbours and friends to call Solihull Social Services and police. But Levi was never taken away from his parents. 'Devastated and disgusted': Levi-Blu's aunt Kirsty Cassin (left) and grandmother Angela Cassin (right) Mr Raggatt said: ‘The totality of the injuries was so severe that they could not have happened by accident, they are the result of at least one very substantial blow to the abdomen and maybe more. ‘The injury was sustained at least six hours, and maybe up to 12 hours, before the 999 call was put in asking for assistance. ‘The defendants are charged jointly with murder because this has to have occurred as a result of cooperation between the two of them. ‘It was a joint enterprise. One person can do the damage, but if the other supports or connives then they are guilty too.’ Following yesterday's verdict, Edwina Grant, chair of the Solihull Local Safeguarding Children Board, said a review was underway. ‘We would like to offer our sincere condolences to the family of Levi-Blu,’ she said. ‘I can confirm Solihull Local Safeguarding Children Board is carrying out a serious case review, the final results of which will be published in the spring.’ | Summary: Levi-Blu Cassin, 22 months, died as result of horrific abdominal injuries. Parents Danielle Cassin & Mark Piper cleared of murder & manslaughter. Jury was unable to pinpoint which one of them had dealt the fatal blows. Sentenced to just nine years in prison at Birmingham Crown Court. It was for causing, or allowing, the baby's death in February last year. Grandmother says family are 'devastated and disgusted' by sentence. | 1,300 | 104 | cnn_dailymail | en |
Summarize: FIELD OF THE INVENTION This invention relates to steel shelving and more particularly to a steel shelving unit providing both adjustable shelving as well as accessory functions for holding items other than on a shelf. BACKGROUND OF THE INVENTION Steel shelving units comprising horizontal shelf-supporting beams with ends adjustably connected to vertical support columns for shelf height adjustment are known. While such units are very useful, it is desirable to provide such beams with improved structures for converting such beams to such columns. As well, it is frequently desirable to provide additional capacity in such units for holding items other than by positioning on the shelves themselves. Additionally, it is desired to provide for vertical adjustability of shelf unit accessories providing such additional capacity. SUMMARY OF THE INVENTION To these ends, a preferred embodiment of the invention includes an improved support column for a shelving unit with provisions for adjustably mounting shelf-supporting beams and accessories, such as a removable, adjustable, hook accessory for hanging items for storage or display, other than on the shelves of the unit. Accordingly, an improved support column according to a preferred embodiment of the invention includes a plurality of perforations or apertures in the column face and which operably accommodate end brackets of a horizontal shelf-supporting beam as well as removable accessories, such as hooks which cooperate with the apertures. Each perforation in the column is defined by a plurality of edges which define tapered support surfaces for cooperating with beam end brackets to adjustably mount the brackets in position up and down the columns. Transverse edges of the perforations or apertures are preferably bent or curved to support a bracket component of an accessory such as a hook. Both the beam-end bracket and the hook bracket interface with two adjacent, vertically-spaced, apertures in the column for securely holding them in a selected vertical position on the column. Preferably the perforations are symmetrical on the column face, and centrally disposed therein so a column can be used on both right and left sides of the shelving unit with all columns in the unit (typically four of them) preferably identical. The beam-end brackets are each angular or L-shaped, with one leg welded or affixed to the beam, and the other leg for lying adjacent to or on the column face over at least part of the column aperture. This other bracket leg preferably has tabs for extending into and engaging a tapered edge of each of two column apertures, one above the other, to hold the bracket and its fixed beam in a fixed, but removable position at selected vertical positions on the column. The hook bracket has extensions, one fitting over a curved portion of an upper aperture on the column and another over a similar curved portion of a next lower aperture for securing the hook in the column face at a selected vertical position. A tab, upwardly extending from the hook bracket, engages a rear side of the column face at the upper aperture to facilitate mounting ad holding the hook and accessory thereon. Other accessories, similarly mountable to the columns, and other than hooks, are contemplated. Accordingly, the invention provides a shelving unit having vertically adjustable shelf-supporting beams, as well as vertically adjustable accessories, both mountable on a supporting column at variable, selected, vertical positions. Improved perforation on the faces of the columns facilitates both beam and accessory mounting. These and other objectives and advantages will become readily apparent from the following detailed description of a preferred embodiment of the invention and from the drawings in which: BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a front isometric view of the overall shelving unit of the invention; FIG. 2 is a front isometric view of a column of the invention; FIG. 3 is a front isometric view of a horizontal shelf-supporting beam of the invention; FIG. 4 is a fragmentary isometric cross-sectional view generally taken along lines 4 - 4 of FIG. 3 ; FIG. 5 is a rear isometric view of the fragmentary isometric cross-sectional view of FIG. 4 ; FIG. 6 is an enlarged isometric view of the encircled area of FIG. 1 ; FIG. 7 is a front partial view of the beam and post of FIG. 6 ; FIG. 8 is a rear view of the partial beam and post of FIG. 7 ; FIG. 9 is a cross-sectional view taken along lines 9 - 9 of FIG. 8 ; FIG. 10 is a front isometric view of a hook accessory spaced from a column shown in partial view; FIG. 11 is an isometric view as in FIG. 10, but showing the hook accessory mounted to the column; FIG. 12 is a cross-sectional view taken along lines 12 - 12 of FIG. 11 but showing the respectively rotated hook accessory being partially mounted to a column; and FIG. 13 is a view similar to FIG. 12 but showing the hook accessory mounted to the column. DETAILED DESCRIPTION OF THE INVENTION Turning now to the drawings, there is shown in FIG. 1 an isometric view of a dual function shelf unit 10 according to one embodiment of the invention. Shelf unit 10 includes four vertical support columns 11 - 14, a plurality of horizontal shelf-supporting beams 15 - 22, a plurality of shelves 23 - 26 which are shown as wire-formed shelves but could be in other wire patterns, of solid material, of grate or other patterns or of any suitable shelf configuration, each supported by one of beams 15 - 18 and one of beams 19 - 22, respectively. A plurality of horizontal braces 27 - 30 extend between respective columns 12, 13 and 11 - 14 as shown, as well as a plurality of slanted braces 31, 32 as shown. Optionally, a plurality of preferably identical tie bars 33, 34, 35 extend between respective ones of the front beams 15 - 18 and rear beams 19 - 22 to support shelves 23 - 26 respectively, these bars 33 - 35 extending respectively under each shelf and supported on the respective horizontal beams, particularly on flange 78 (see FIGS. 4-6 ). A column 12 is illustrated in FIG. 2, column 12 being preferably identical to columns 11, 13, 14 and thus only one being described in detail, each with preferably like parts. Identical construction allows a column to be used at any corner position in unit 10. Columns 11 - 14 are in a C-shaped cross-sectional configuration (see also FIGS. 6, 7, 8, 10 - 13 ). Each column thus has a face 40, a plurality of symmetrical apertures 42 in the face 40, and a plurality of rectangularly-shaped apertures 44 in each leg 46, 48. Each leg terminates along its legs 46 - 48 with its flanges 50, 52 completing configuration of column 12 (see FIGS. 2, 6 and 9, for example). Details of apertures 42 are perhaps best seen in FIGS. 6-13. Referring first to FIG. 6, each aperture 42 is preferably identical to each other aperture 42 in the respective columns. Each aperture 42 is oriented one above the other in the columns, equally spaced vertically and aligned centrally and symmetrically in column face 40, as shown. Each aperture is defined in part by edges 54, 56, tapering or inclined from a bottom edge 58, 60 upwardly and outwardly in face 40 to upper edges 62, 64. A curved tab 66 extends upwardly between bottom edges 58, 60 and is turned or curved inwardly from face 40. A second tab 68 extends downwardly as part of face 40 between edges 54, 56 and has an inwardly curved tab end 70, as shown. Tapering or inclined edges 54, 56 may be continuously straight, or may define slightly different angles, such as at breaks 72, 74 as illustrated in FIG. 6 such that upper regions of edges 54, 56 incline outwardly in face 40 at a slightly greater angle than lower regions of those edges to facilitate initial bracket engagement. Horizontal beams 15 - 22 are, like columns 11 - 14, each preferably identical to each other so they can be interchangeably used in shelf unit 10. Only one exemplary beam 17 will be described in detail. With initial reference to FIGS. 3-5, an elongated shelf support beam 17 includes a preferably embossed face 76, having a plurality of outstanding ornamental projections 77 of any suitable form embossed, pressed from or otherwise defined in face 76. Beam 17 is defined by a lower, rearwardly extending flange 78, a first rearward extending upper flange 79, a return 80 and a final rearwardly extending shelf-supporting flange 81. Other beam configurations may be used but this beam provides both shelf-supporting flange 81 and lower, tie-bar supporting flange 78 as will be described. To an end of beam 17 is welded (or otherwise fixed) an L-shaped bracket 82 having a rearwardly extending leg 83 and a front leg 84. A bracket 82 a, which is essentially a mirror image and otherwise identical to bracket 82, is fixed to the other end of beam 17 and is otherwise similar to the bracket 82. Similar parts of the respective brackets are numbered with a suffix “a”. Legs or faces 84, 84 a of brackets 82, 82 a are provided with punched-out upper and lower locking tabs 87, 88, 87 a, 88 a formed at an angle in leg 84, 84 a, respectively ( FIGS. 4, 5 ). As will be described, these tabs serve to engage and interface with inclined edges 54 of adjacent apertures 42 (see FIGS. 8, 9 ) holding and locking the brackets 82 to column 12. Similar tabs 87 a, 88 a will engage and interfere in similar fashion with inclined edge 56 of apertures 42 in another column 11 at the other end of the beam to hold and lock the bracket 82 a and beam 17 to that column 11. FIGS. 6-8 further illustrate the interconnected relationship of a beam 17 to a column 12. The other beam end is supported and locked such as on a column 11 by a bracket 82 a operational in a similar fashion. In FIG. 6, bracket 82, via tabs 87, 88 is locked onto column 12. For example, the beam 17 and its fixed bracket are oriented proximate vertically adjacent apertures 42 in column 12, with tabs 87, 88 initially proximate upper ends of apertures 42 so the tabs can be inserted into the apertures. When leg 82 is against face 40, the bracket (and beam) is moved downwardly, tabs 87, 88 engaging and fitting around inclined edges 54 in the respective apertures. The distance between tabs 87, 88 and the leg 82 of the bracket causes, as the bracket moves downwardly, the tabs 87, 88 and leg 84 to be frictionally wedged onto column 12, the engagement of respective tabs 87, 88 with edges 54 providing frictional, locking engagement of bracket 82 and beam 17 to column 12. Similar complementary action of tabs 87 a and 88 a with inclined edges 56 of vertically-adjacent apertures 42 in column 11 secures the other end of the beam in vertically-coordinated position so the beam 17 is horizontally supported across and between columns 11 and 12 as described. The vertical locations of the beams 15 - 22 can be set on the columns as desired to provide the eventually desired spacing between any shelves as described in unit 10. It will be further appreciated that the frictional interface between two respective apertures 42 in each column, and the single complimentary brackets at each end of the beam strengthens and rigidifies any tendency of the columns connected by the beams to “rack”, move or tilt toward or away from one another, resulting in a very strong, rigid unit 10 construction. This benefit is, in part, also provided by the engagement of the inner faces of legs 83, 84 with the respective leg 46 and face 40 of column 12 as well as tabs 87, 88 and inclined aperture edge 54. Complementary engagement of complementary parts of bracket and column at the other beam end provide the same result. Accordingly, it will be appreciated that a shelf unit 10, as described above, provides a rigid shelving function for a variety of applications. In a further embodiment of the invention, a further support function is provided by the addition of an accessory which provides a further article support or hanging function shelving unit 10. This additional embodiment is illustrated in FIGS. 10-13 of the drawings, wherein an accessory hook 90 is provided for use on a column, such as a column 12 as described above. In FIG. 10, hook accessory 90 includes a hook member 91 preferably rigidly-mounted at shank 91 a to a hook bracket 92. Bracket 92 includes a face surface 93, a lower flange 94 with a depending tab 95, and upper flange 96 with a downwardly depending tab 97, and an upwardly extending locking tab 98. FIG. 10 illustrates a column 12 and a hook accessory 90, not yet assembled and FIG. 11 illustrates a hook accessory 90 attached to a column 12. In FIG. 11, it is noted that flanges 94 and 96 of hook accessory 90 rest on the curved tabs 66 respectively of vertically-oriented adjacent apertures 42 in face 40 of column 12. Tabs 97, 95 prevent hook accessory 90 from being pulled outwardly, away from column 12. Locking tab 98 prevents removal of hook accessory 90 horizontally and forwardly of column 12 since it engages or is in close blocking proximity to curved end 70 of tab 68 if moved directly forwardly. Referring to FIGS. 12 and 13, FIG. 12 illustrates the preferred motion for attaching accessory hook 90 to a column such as 12. In use, the upper end of hook 90 is inserted into an upper aperture 42, tab 98 extending therein below, then behind end 70 of tab 68 in the aperture. Once the upper end of accessory hook 90 is extended into upper aperture 42, the lower end of bracket 92 can be rotated into a lower aperture 42 and then bracket 92 lowered so tabs 97, 95 are locked behind the curved tab ends 66, respectively of the vertically-adjacent apertures as in the cross-sectional view of FIG. 13. Hook accessory 90 is thus removably but securely mounted at selected vertical positions up and down column 11, 12 and others, providing for additionally supporting functions for a variety of items on shelving unit 10. In this embodiment, the hooks can be selectively spaced along the entire lengths of the columns excepting at the same position of the brackets 82, 82 a on the columns for the horizontal shelf-supporting beams. Vertically-adjustable storage is thus not limited to the shelves only but includes the additional function of hanging items suitably on vertically-adjustable hooks. It will be appreciated that other storage or hanging accessories can be similarly attached to the columns to provide additional hanging or storage functions. | Summary: A dual function shelf unit has identical support columns, horizontal shelf-supporting beams adjustably-mounted on the columns for vertical shelf adjustment and one or more hooks also mountable on at least one column. The columns have apertures with complementary apertures having opposed inclined edges for accepting a beam-end bracket from either side of the column and tabs for accepting mounting flanges of hooks. Both beam-end brackets and hooks are each mounted in two vertically-spaced apertures. Vertically adjustable shelf and hook storage is provided. | 4,009 | 129 | big_patent | en |
Summarize: A horrific video has emerged showing puppies and kittens panicking as they wait to be dissected at an animal testing laboratory. The footage was filmed as part of an undercover investigation by the British Union for the Abolition of Vivisection (BUAV) at the government-licensed centre. It shows the animals - some as young as four-weeks-old - screaming, crying and whimpering as they are taken from their mothers and killed. WARNING: GRAPHIC CONTENT. Scroll down for video. Horrific: A video, filmed by an undercover investigator, shows puppies and kittens panicking as they wait to be dissected at an animal testing laboratory. Above, a researcher prepares to cut into a puppy. Awaiting their fate: The footage, captured as part of an investigation by the British Union for the Abolition of Vivisection (BUAV), shows the animals screaming and crying as they are taken from their mothers and killed. Sad: It also features puppies and kittens - some as young as four-weeks-old - shrieking in terror as they are restrained by researchers - just seconds before they are given a lethal injection and cut up for examination. The video was taken by an investigator, known as 'Susie', who worked at the laboratory for eight months until December last year, according to the Sunday Express. It shows puppies and kittens shrieking in terror as they are restrained by researchers - just seconds before they are given a lethal injection and cut up for examination. Shockingly, their dissected bodies are later dumped in a bin as waste. In the video, an employee can be seen disposing of a kitten's body - saying: 'That's you done, you can go in our bin'. Cuddled up: The video was taken by an investigator, known as 'Susie', who worked at the laboratory for eight months until December last year. Above, three puppies curl up, unaware that they will soon be used in tests. Examination: After the young animals are killed and dissected, their bodies are dumped in a bin as waste. Meanwhile, a black puppy can be heard. screaming and thrashing around as two researchers prepare to take its. blood at the centre, which is owned by US pharmaceutrical giant Merck. Sharp & Dohme (MSD). And a young canine yelps loudly. as a female employee struggles to find a suitable vein in his leg in which to inject him. 'Oh. dear. What a fuss, what a fuss,' the woman can be heard saying. 'Perhaps. your leg was a bit sore. Was it from all those bleeds? Oh dear, oh. dear.' The employee eventually administers the lethal dose after trying the puppy's other leg - before commenting: 'That's gotcha.' Family: Many of the animals are born at the laboratory for the sole purpose of being dissected for examination at just a few weeks old - with their healthy mothers (including this dog, above) often killed just hours later. She then adds: 'Put up a bit of a fight, didn't you, yeah?' The footage also shows scenes of puppies and kittens lying on operating tables as employees insert needles and cutting equipment into their bodies. In one particularly horrific scene, a researcher lifts a motionless puppy onto a table, saying: 'Yes,. this is where it gets messy. Right, OK. Come on then little fella. 'Let's make sure you have departed before we start getting our bits and pieces.' A staggering 92 puppies, 10 adult female dogs and at least 15 kittens were killed during the investigation at the centre, according to BUAV. Waiting to die: A staggering 92 puppies, 10 female dogs and at least 15 kittens were killed during the project. Eating: Some had reportedly been imported into the UK from overseas breeding farms at just a few weeks old - causing them to suffer from ulcers, high temperatures and weight loss. Above, dinner time at the centre. However, the undercover investigator managed to rescue two adult dogs, Bonnie and Billie, and a five-month-old puppy, Oliver, during the project - while two others were rehomed. Many of the animals are believed to have been born at the laboratory for the sole purpose of being dissected for examination at a young age - with their healthy mothers often killed just hours later. Others had reportedly been imported into the UK from overseas breeding farms at only a few weeks old - causing them to suffer from ulcers, high temperatures and weight loss. Action: Home Office minister, Norman Baker (above), is believed to have called for a full report into the laboratory's activities. During the eight-month period, the facility also dissected a number of rabbits and chickens, according to BUAV. Today, Michelle Thew, chief executive of BUAV, said the union had released the video in a bid to add transparency to the public debate about animal testing. 'This is a secret the research industry would never want to be. released into the public domain,' she said. 'Millions of families throughout the UK. who share their homes and lives with cats and dogs will be appalled by. these revelations. 'It is unacceptable, not only that these animals are. suffering and dying in this way, but that many of them could have been. released into loving homes instead of being killed and discarded for. convenience sake.' She added that thousands of dogs and hundreds of cats are used in research every year in UK, despite widespread concern about their use. Meanwhile, Home Office minister, Norman Baker, told the Sunday Express his department had carried out 'a number' of inspections at the. facility over the past year - but added that he has now called for a full report into the centre's activities. MSD told MailOnline that it 'adheres to all regulatory standards of testing and development of. vaccines'. It added that it is obliged by law to test certain drugs on animals, including vaccines for kennel cough, parvovirus and feline calicivirus. A spokesman said: 'Animal well-being is core to our mission. All MSD animal health research is performed by qualified, trained personnel. 'Our facilities are in full compliance with all laws and regulations, and procedures and facilities are regularly reviewed and inspected by relevant regulatory authorities.' | Summary: Footage shows dozens of puppies and kittens taken from mothers and killed. They can be heard screaming in terror as they are restrained by researchers. They are later killed and dissected, before bodies are dumped in bin as waste. One employee can be heard saying 'That's you done, you can go in our bin' Animals are as young as four-weeks-old - and their mothers are also killed. Video filmed by investigator working undercover at facility for eight months. Employee was working on behalf of British Union for Abolition of Vivisection. Lab owned by pharmaceutical giant Merck Sharp & Dohme. MSD says it did not break any laws and adheres to animal testing regulation. | 1,479 | 156 | cnn_dailymail | en |
Summarize: By. Helen Pow. and Associated Press. PUBLISHED:. 11:28 EST, 25 February 2014. |. UPDATED:. 12:56 EST, 25 February 2014. Penance: Eric Justin Toth, pictured, was captured in Nicaragua after a year on the FBI's 'Ten Most Wanted' fugitives. A Washington private school teacher captured in Nicaragua after a year on the FBI's 'Ten Most Wanted' fugitives list said he knew from age 13 that he was attracted to young boys and that his urges were 'dangerous' in a letter to the judge tasked with sentencing his child pornography case. Eric Justin Toth told the judge he plans to spend the rest of his life 'doing penance' for his crimes and said he wants to spend his time in prison 'as productively as possible,' including participating in a program to train seeing eye dogs. Toth fled Washington in 2008 after images. of child pornography were found on a camera he had used while a teacher. at Beauvoir, a private elementary school located on the grounds of the. Washington National Cathedral. The FBI added Toth to its Most Wanted list in 2012, where he filled a vacancy created by Osama bin Laden's death. He was finally caught last year in Nicaragua after five years on the run. In the two-page letter that was submitted to federal court Monday ahead of his March 11 sentencing, Toth acknowledged that he became aware of his attraction to boys when he was just 13. 'I knew society considered people like me monsters, so I swore myself to secrecy,' he wrote. He went on to apologize for his actions and said he has always known his urges were 'dangerous.' 'For. years, my brain manufactured one justification after another for giving. into my sexual urges. In more recent years, I've worked hard to change. that and continue to work on it in prison. In my heart of hearts, I've. always known my urges are dangerous,' he wrote. Toth's lawyers are recommending that he spend 22 years in prison. That's the low end of a recommended sentencing range agreed to by Toth as part of a guilty plea last year. Most Wanted: The FBI added Toth to its Most Wanted list in 2012, where he filled a vacancy created by Osama bin Laden's death. Toth's lawyers said he has no criminal. record, a history of psychological problems including depression and. that when he was in high school he was sexually abused by a teacher. Prosecutors. asked that Toth be sentenced to 30 years in prison. Both groups of. lawyers agreed that after prison he should spend a lifetime on. supervised release. Toth's lawyers wrote that he would like to serve his sentence at a New Jersey prison that has a program to train the guide dogs. Toth ended his letter by saying he. will 'commit the rest of my life to doing penance, to kindness and. decency, and to honest and perpetual accountability.' Toth. pleaded guilty in December to three counts of producing child. pornography, identity theft and misuse of a Social Security number. He. acknowledged that in 2005, while working as a counselor at a Wisconsin,. he took photographs and videos of a sleeping male camper. School: Toth fled Washington in 2008 after images of child pornography were found on a camera he had used while a teacher at Beauvoir, pictured, a private elementary school located on the grounds of the Washington National Cathedral. In 2006, after he began working as a third-grade teacher at Beauvoir, he took pictures and videos of one student. He also installed a hidden video camera disguised as an air freshener in a school bathroom. The camera captured 15 children using the bathroom. Toth was escorted off Beauvoir's campus in June 2008 after school administrators found images of child pornography on a school camera he used. A media card containing more images was found at the school soon after, but Toth fled before police could arrest him. He spent time in Texas and Arizona before authorities caught him in Nicaragua | Summary: Eric Justin Toth, formerly a teacher at Beauvoir elementary school in Washington, wrote a two-page letter to the judge tasked with sentencing him on March 11. Toth fled Washington in 2008 after images of child pornography were found on a camera he had used while at the private school. The FBI added Toth to its Most Wanted list in 2012, where he filled a vacancy created by Osama bin Laden's death. Toth, who was captured last year in Nicaragua after five years on the run, told the judge in his letter that he hopes to spend his time in prison 'as productively as possible' | 993 | 145 | cnn_dailymail | en |
Summarize: This application is continuation of application Ser. No. 08/135,575, filed Oct. 13, 1993, now abandoned which is a continutation of application Ser. No. 08/022,433, filed Feb. 16, 1993, now abandoned which is a continuation of Ser. No. 07/540,399, filed Jun. 18, 1990, now abandoned which is a continuation of Ser. No. 07/279,907, filed Dec. 5, 1988, now abandoned. The present invention relates to improved methods of food processing. More specifically it relates to improving the firmness of fruits and vegetables which are processed by blanching followed by sterilization. BACKGROUND OF THE INVENTION Thermal processing is one of the most important methods mankind has developed for extending the storage life of perishable foodstuffs. However, the thermal process causes some destruction of the food qualities. Nutritional value, texture, color and flavor are usually damaged to a greater or lesser extent during the thermal process. The soft texture of most canned vegetables is recognized as a major quality defect. It is probably one of the main reasons why the sales of canned vegetables are declining while sales of fresh vegetables are increasing. Protecting food against excessive softening caused by thermal processing is an on-going problem. VanBuren et al, J. Food Sci., 27:291 (1962), have processed snap beans using a low temperature blanch at about 170° F. before canning to give a firmer-textured canned snap bean as compared to the conventional blanching at 200° to 212° F. The vegetable canning industry typically uses a blanch temperature of about 170° F. for snap beans. Similarly, Lee et al, J. Food Sci., 44:615 (1979) have shown that a 170° F. blanch gives firmer textured canned carrots. Large quantities of vegetables and fruits are preserved by canning. This technology requires enclosing the product in hermetically sealed containers and heating at a specified temperature for a specified time to destroy all microorganisms inside the container. Products with a pH above 4.5 require a substantial heating regime to obtain commercial sterility. For example, green beans packed in brine in 1 lb cans require 22 minutes at 240° F. or 13 minutes at 250° F. This heavy heat treatment cannot be compromised because microorganisms of public health significance, such as Clostridium botulinum, require this degree of heat treatment to be destroyed. Unfortunately, this amount of heat causes great damage to the food texture. Most canned vegetables have a softer than desirable texture. The present invention relates to a process for improving fruit and vegetable firmness, particularly in thermal processed foods. BRIEF DESCRIPTION OF THE INVENTION One object of the present invention is to improve the firmness of canned fruits and vegetables subjected to thermal sterilization processing by predetermining optimum low-temperature blanch temperatures for each food from softening curves and a plot of the rate of firmness increase with blanch temperature. Another object is to provide a process to prepare canned fruits and vegetables having improved firmness qualities by using lower blanch temperatures and optimum holding times prior to sterilization to replace or as an adjunct to conventional processing methods. Another object is to maximize the factors that contribute to food firmness by selecting conditions for increasing the firmness and simultaneously countering conditions which have the effect of softening the food. Yet another object relates to a low temperature blanching process for maintaining the firmness of fruits and vegetables subjected to sterilization processing which comprises blanching the fruits or vegetables at a temperature of from about 125° F. to about 160° F. prior to sterilization for a time sufficient to cause said fruit or vegetable to remain firmer after sterilization as compared to said fruit or vegetable sterilized without said blanching step. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a softening curve graph of log extrusion force (KN) versus process time in minutes for diced beets processed at 220° F. FIG. 2 is a softening point curve where firmness (N) is plotted against processing time in minutes for cut green beans in #303 cans, and blanching is carried out for at 3 minutes at 74° C. (165.2° F.); the National Food Processors Association recommends a process time of 22 minutes at this temperature. FIG. 3 is a graph comparing softening curves for diced Nantes carrot blanched 4 minutes at 74° C. and diced Nantes carrots blanched 4 minutes at 100° C. respectively before processing at 100° C. where firmness (kN) is plotted against processing time in minutes. FIGS. 4(a)-4(g) are referred to in Example 1 and are a series of graphs for Nantes carrots processed at 250° F. where the log of the firmness is plotted against process time in minutes together with equations for lines of best fit and correlation coefficient R, where the data is used to measure substrate "b" and thermal firmness value (y-axis intersect). FIG. 5 is a firmness increase overview for Chanteney variety carrots and depicts a plot of the thermal firmness versus hold time (minutes) after 3 minutes blanching at various temperatures where the slope of lines equal the rate of firmness increase. FIG. 6 is a plot of the rate of firmness increase (Newtons per minute) vs. blanch temperature (degrees Fahrenheit) for Chantenay var. carrots. From the graph, optimum blanch temperatures can be selected based on rates of firmness increase. FIG. 7 is a graph of rate of firmness increase versus blanch temperature for three varities of green beans, namely Labrador, Bonanza and BBL 47. FIG. 8 is a graph of rate of firmness increase versus blanch temperature for three varieties of carrots, namely Nantes, Danvers and Chantenay. DETAILED DESCRIPTION OF THE INVENTION The invention relates to improved methods of food processing and particularly relates to improving the firmness of fruits and vegetables over commercial processes requiring a blanching step followed by sterilization. The invention relates to a process to increase or maintain the firmness of fruits and vegetables subjected to thermal sterilization processing which comprises; a) preparing a fruit or vegetable for sterilization processing; b) blanching the fruit or vegetable at a temperature in the range of from about 125° F. to about 160° F., preferably from 135° F. to 155° F. and most preferably at temperatures from about 140° F. to about 150° F.; wherein the optimum blanching temperature is determined in advance for the fruit or vegetable as follows: (1) determining the thermal firmness of the said fruit or vegetable as a function of the blanching temperature and blanch time; (2) determining the rate of thermal firmness increase with blanch time for a series of blanching temperatures; (3) selecting blanch temperatures or ranges thereof based on (1) and (2) adapted to provide an optimum firmness increase. c) holding the blanched fruit or vegetable for a hold period of time up to 120 minutes before sterilization processing; d) sterilizing the product resulting from step (c). The thermal firmness and the rate of increase of thermal firmness with blanch temperatures and blanch time can be determined by various methods. A preferred method is to determine the firmness of the fruit or vegetable as a function of the blanching temperature and blanch time and determine the thermal firmness therefrom. By plotting the thermal firmness versus blanch time for a series of blanching temperatures, one determines the rate of thermal firmness increase. When the rate of thermal firmness increase is graphed against blanch temperature a curve is obtained which allows one to conveniently select a blanch temperature adapted to provide an optimum firmness increase. Another aspect of the invention relates to a method for further enhancing the firmness of fruits or vegetables subjected to thermal processing by combining the above process with treatments using food-grade multivalent salt compounds such as for example magnesium chloride, magnesium oxide, magnesium sulfate, calcium chloride, calcium sulphate, calcium oxide, calcium acetate, calcium citrate and the like; food grade acids selected from the group consisting of citric acid, acetic acid, malic acid, tartaric acid, latic acid and the like or combinations of both. It is recognized that the invention can be practiced using a variety of blanch temperature and times preceding further processing steps including thermal sterilization. Thus blanch time as used herein is defined broadly as the time the product is held at the blanch temperature and includes a holding time where the blanched product is held for a defined period prior to sterilization processing. Another aspect of the invention relates to the use of a low blanching temperature sufficient to activate the natural enzyme which promotes firmness in said fruit or vegetable but lower than the temperature which inactivates such enzyme. DISCUSSION Kinetic studies have shown that the rates of thermal softening of thermally processed fruits and vegetables is a two-phase process. There is an initial rapid rate of softening which is followed by a much slower rate of softening. Huang and Bourne (J. Texture Studies, 10:1-23 (1983)) investigated the effect of thermal processing on the firmness of vegetables. These authors measured firmness by placing the sample in a back extrusion cell mounted in the Instron Universal testing machine (cf. Bourne and Moyer 1968). The back extrusion cell used was 10.2 cm I.D. by 12 cm height with a 4 mm annulus. Extrusion speed was 30 cm/min and the downward movement of the plunger was reversed 6 mm from the bottom of the cell. The maximum peak of the recorded force-distance curve, measured in Kilonewtons, was taken as the firmness of the commodity. The effect of process time on firmness of fruits and vegetables can be shown by plotting log extrusion force vs. process time. Typical softening curves are shown in FIGS. 1, 2 and 3. The softening curve is characterized by an initial rapid decrease in firmness (negative slope) that is almost linear but which curves off into a second straight line with a shallow negative slope at longer process times. Since a first-order kinetic process is represented by a rectilinear plot on a semilogarithmic scale it is evident that simple first-order kinetics does not apply when lengthy process times are used on canned vegetables. The general shape of this curve is typical for all vegetables studied. Fruits show similar thermal softening curves but the initial rate of softening is completed more rapidly than for vegetables. The shape of these experimental curves is similar to that obtained for the sum of two independent simultaneous first-order processes occurring at different rates. From analogy with kinetic theory the linear portion of the semi-logarithmic curve that is obtained after prolonged heating times (referred to as mechanism 2) gives the apparent softening rate constant for this mechanism. When the linear portion of mechanism 2 is extrapolated back to zero process time and the extrapolated line subtracted from the line above it, the result is a second straight line with a much steeper slope (referred to as mechanism 1) and the slope of this derived line gives the apparent softening rate constant for mechanism 1. This kinetic evidence indicates that the softening of vegetables during thermal processing is composed of two pseudo first-order processes with different rate constants occurring simultaneously. One process is rapid and the other process is slow. From analogy with kinetic theory for two apparent first order processes we can postulate that the firmness of vegetable tissue is composed of two substrates, "a" and "b" and that substrate "a" softens rapidly by mechanism 1 while substrate "b" softens slowly by a different mechanism (mechanism 2). When the linear portion of mechanism 2 is extrapolated back to zero process time (dotted line in FIG. 1) and this extrapolated line is subtracted from the solid line above it, a second line is obtained as shown by the open circles and dashed line in FIG. 1. The derived dashed line represents mechanism 1 and the slope is its apparent rate constant. The linear portion of the solid line represents mechanism 2 and the slope is its apparent rate constant. Predicting Process Conditions for Optimum Firmness As shown in the best mode examples (Table 1, FIG. 6), a plot of the rate of firmness increase (Newtons per minute) versus blanch temperature gives a graph which allows one to select blanch temperatures to produce optimum firmness in the shortest time. For example, with reference to FIG. 6, suitable blanch temperature ranges for Chantenay variety of carrots can be selected directly from the graph. Broad ranges are those falling within rate of firmness increase (Newtons/minute) of up to about 1.5, i.e. temperatures of 125° F. to 170° F.; preferred ranges are those with rate of firmness increase of 1.50 or greater (i.e. temperatures of from about 135°-155° F.) and most preferred ranges are those having positive rate increase of 2.5-3.0 (i.e. temperatures of from about 140° to about 150° F.). It is recognized that because of the differences in the food types to be processed, the temperatures and the holding times sufficient to produce the described rate of firmness increase will vary. Generally the best blanching temperature will be broadly in the range of about 120° F. to 160° F. sufficient to provide a fast rate of firmness increase in Newton's/minute; preferably at about 135° F. to about 155° F.; and most preferably at a temperature from about 140° to about 150° C. sufficient to provide the fastest rate of firmness rate increase. It is recommended that experiments be run to establish graphs for each food type to be processed. A wide variety of vegetables can be processed according to the present invention. These include for example, carrots, beets, potatoes, wax beans, green beans, cauliflower and the like. Similarly the invention is applicable to a wide variety of fruits such as peaches, apples, cherries, pears and the like. The following best mode examples are meant to illustrate the invention; they should not be narrowly constructed as to limit the invention. EXAMPLE 1 Two carrot varieties (Nantes and Chantenay) were washed, topped, diced into 3/8" cubes on an Urschel dicing machine and small pieces removed by passing over a shaking screen. Seven 5 Kg lots of diced carrot were weighed for each blanch temperature. Nine blanch temperatures were used--120°, 130°, 140°, 150°, 160°, 170°, 180°, 190°, 200° F. Each lot was blanched 6 min. in water at the designated temperature, then removed from the water and held with no further heating for a designated hold time, then blanched again 3 minutes at 212° F. in water to stop further enzyme activity and cooled by immersion in cold water. Hold temperatures were 0, 15, 30, 45, 60, 75, 90 minutes. Sixteen #303 cans were filled from each of the 63 blanch temperature-hold time combinations (7 hold times×9 blanch temperatures). One 60 grain salt tablet was added to each can. The cans were filled with near boiling water, closed, and processed at 250° F. in steam in still retorts with pressure cooling at the conclusion of the designated process time. For each blanch temperature-hold time: 4 cans were processed 40 minutes, 4 cans were processed 60 minutes; 4 cans were processed 80 minutes; 4 cans were processed 100 minutes. Beginning one week later, the cans were opened and the firmness measured using a back extrusion cell (7.4 cm I.D.×7.8 cm internal height with a 4 mm wide annulus) mounted in an Instron Universal testing machine. This machine plots on a strip chart the force required to extrude the vegetable up through the 4 mm wide annulus between the descending ram and the inside wall of back extrusion cell. This test was replicated eight times for each sample. The maximum force was measured from the Instron chart and the mean value calculated for each treatment. The logarithm of the mean firmness was graphed against the process time in minutes. A typical series of graphs so obtained is shown in FIGS. 4(a) -4(g) which give the data for the 140° F. blanch treatment of Chantenay carrots. The calculated line of best fit to the data points is drawn. The intercept of this line on the vertical axis (firmness at zero process time) is called "thermal firmness". The thermal firmness data are then graphed against hold time after blanch and the calculated time of best fit is drawn. A typical graph is shown in FIG. 5 for each of 9 blanch temperatures for Chantenay carrot. The slope of each line is measured and this is the rate of increase in thermal firmness for each blanch temperature (shown at the right hand edge of the figure). The data obtained from FIG. 5 is then graphed against blanch temperature to give FIG. 6. This figure shows that the rate of increase of thermal firmness increases as the blanch temperature rises from 120° F. to 150° F. and then decreases at temperatures above 150° F. In this case, for Chantenay carrot, 150° F. blanch gives the fastest rate of increase in thermal firmness. Food processors prefer to keep holding times as short as possible. Graphs like FIG. 6 enable processors to determine a blanch temperature-hold time regime that will give a firmer textured product in the shortest possible time. Table 1 shows thermal firmness values versus hold times in minutes for Chantenay and Nantes variety carrots. TABLE 1______________________________________Thermal Firmness (Force (N)) Versus Hold Time*______________________________________Chantenay CarrotsHold Time Blanch Temperatures (°F.)(minutes) 120 130 140 150 160 170______________________________________0 199.5 195.0 245.5 239.9 251.2 223.915 182.0 234.4 346.7 363.1 269.2 234.430 218.8 316.2 371.5 436.5 263.0 218.845 234.4 316.2 501.2 467.7 302.0 245.560 213.8 323.6 501.2 457.1 346.7 245.575 229.1 309.0 467.7 524.8 371.5 229.190 281.8 331.1 489.8 549.5 389.0 234.4______________________________________Chantenay CarrotsHold Time Blanch Temperatures (°F.)(minutes) 180 190 200______________________________________0 213.8 204.2 218.815 213.8 204.2 195.030 223.9 213.8 213.845 234.4 229.1 223.960 218.8 234.4 199.575 229.1 199.5 204.290 218.8 199.5 195.0______________________________________Nantes CarrotsHold Time Blanch Temperatures (°F.)(minutes) 120 130 140 150 160 170______________________________________0 169.8 195.0 169.8 204.2 269.2 245.515 269.1 208.9 239.9 309.0 302.0 263.030 275.4 218.7 269.2 346.7 363.1 263.045 281.8 295.1 316.2 380.2 398.1 257.060 338.8 295.1 363.1 446.7 380.2 302.075 309.0 309.0 338.8 457.1 436.5 263.090 426.6 380.2 398.1 467.7 426.6 218.8______________________________________Nantes CarrotsHold Time Blanch Temperatures (°F.)(minutes) 180 190 200______________________________________0 223.9 173.8 166.015 186.2 169.8 173.830 213.8 182.0 169.845 229.1 166.0 154.960 204.2 166.0 169.875 213.8 182.0 162.290 223.9 173.8 162.2______________________________________ *Time Interval Minutes between end of blanch and beginning of thermal sterilization. Table 1 shows that a conventional process of a 200° F. blanch (no hold time) give thermal firmness values of 219N and 166N respectively for Chantenay and Nantes variety carrots with no increase of firmness for holding times of 30 minutes after blanching. However, when the carrots are blanched at 150° F. the respective thermal firmness values are 240N and 204N and these increase to 437N and 346N at 30 minutes hold and to 550N and 460N at 90 minutes hold time. Thus a low temperature blanch temperature plus hold time before sterilization gives a marked increase in firmness. FIG. 5 shows a plot of thermal firmness versus hold time (minutes) for Chantenay carrots. It is seen that firmness increases most rapidly for 150° F. and 140° F. blanched carrots, less rapidly for 130° and 160° blanch. There is only slight increase of thermal firmness with hold time for the remaining blanch temperatures. FIG. 6 is a graph of rate of firmness increase (newtons/minute) versus blanch temperature for Chantenay carrots. Both FIGS. 5 and 6 allow one to select optimum blanch temperatures and hold times for optimum firmness. Referring to FIG. 6 it is seen that desirable blanch temperatures for Chantenay carrot are broadly from about 125° F. to about 160° F., preferably from about 135° to 155° F. and most preferably from about 140° to about 150° F. EXAMPLE 2 In an experiment similar to that of Example 1, Danvers variety of carrots was evaluated using 4° F. increments in blanch temperatures over the range of 140° to 160° F. to more critically define this range. The results are shown in Table 2 and FIG. 8. TABLE 2______________________________________Firmness of Canned Denvers Carrot (Newtons Force)Hold Time Blanch Temperatures (°F.)(minutes) 140 144 148 152 156 160______________________________________0 247.6 206.0 191.0 234.9 259.2 254.315 294.6 333.3 303.2 339.0 337.5 264.730 364.6 327.2 376.5 368.2 359.5 270.745 452.7 444.6 393.7 402.1 394.7 287.460 513.7 452.6 413.0 405.7 352.7 299.675 424.1 449.4 400.5 432.4 372.2 293.390 449.1 474.2 437.3 393.2 364.5 304.1______________________________________ From the above it is seen that the 144° F. blanch temperature gave the fastest rate of increase in firmness; however, temperatures of 140°, 148° and 152° F. also gave high values for the rate of firmness increase. FIG. 8 also shows a rate of firmness increase for two other carrot varieties (Nantes, Chantenay) in the narrower blanch temperature rante of 140° F. to 160° F. EXAMPLE 3 Ten Kg lots of Danvers variety carrots (unpeeled) were blanched 15 minutes and 30 minutes at 150° F. They were then peeled, sliced and diced and sterilized for 24 minutes at 250° F. in #303 cans (21 cans per treatment). For comparison purposes a control was blanched 4 minutes at 212° F. with no hold time before peeling, cutting and sterilization. The firmness results (newton's) are shown in Table 3. TABLE 3______________________________________Firmness of Canned Carrot (in Newton's Force)TreatmentBlanch Temp, °F. Type Firmness(N) & SD______________________________________4 min. at 212° F. (control) slices 214N +/- 11.715 min. at 150° F. slices 255N +/- 10.730 min. at 150° F. slices 328N +/- 14.98 min. at 212° F. dices 202N +/- 8.115 min. at 150° F. dices 268N +/- 10.930 min. at 150° F. dices 317N +/- 15.3______________________________________ From Table 3, it is seen that a 150° F. blanch for 15 and 30 minutes gives a marked increase in firmness as compared with conventional processing of 212° F. for 4 minutes. EXAMPLE 4 In an experiment similar to Example 1, three varieties of snap beans were processed to establish the blanch temperature that gives the fastest rate of increase of thermal firmness. The varieties were Labrador (a green bean), BBL-47 (a green bean) and Bonanza (a wax bean). Example 1 protocol was used except for the following differences: 1) the beans were cut into 1 1/2 inch lengths; 2) hold times of 0, 15, 30, 45 and 60 minutes were used; 3) blanch temperatures were 120°, 130°, 140°, 145°, 150°, 160°, 170° and 180° F. The results are shown graphically in FIG. 7 which is a graph of the rate of firmness increase versus blanch temperature. The difference in the maximum rate of firmness increase is noted i.e. 130° F. for Bonanza; 145° F. for BBL-47 and 150° F. for Labrador. EXAMPLE 5 Heads of cauliflower were broken apart into curds, then blanched 10 minutes at 145° F. in hot water containing 6 grams of citric acid per liter, hold for 20 minutes, then sterilized in No. 303 cans for 22 minutes at 240° F. As a control some of the curds were blanched 4 minutes at 200-210° F. then sterilized in the same manner with no hold time. The mean value of 8 replicate texture measurements showed that the firmness (newton's force) for the 145° F. blanched product was 127 as opposed to 65 for the control. EXAMPLE 6 Small white potatoes (B grade) were peeled in an abrasive rotary peeling machine, then blanched 15 or 30 minutes at 145° F., filled into No. 303 cans and sterilized 26 minutes at 250° F. in a still retort. A control batch of potatoes was sterilized at 250° F. for 26 minutes without blanching. The blanched potatoes gave a mean firmness values of 405 and 431N as opposed to 378N for the control. EXAMPLE 7 Two types of sweet cherries (Sodus-light variety and Duron II - a dark variety) were blanched 5 minutes at 140° F., then held for 30, 60, 120 minutes before being canned in 20 percent sugar syrup and sterilized at 212° F. for 20 minutes. For controls, some cherries were canned with no blanch treatment which is the conventional commercial procedure. Firmness was measured as the force in newtons to push Dunkley cherry pitters simultaneously through 30 cherries. The firmness is shown in Table 4. TABLE 4______________________________________ Firmness Hold Time (Newtons Force)Treatment, Blanch Temp. °F. (minutes) Sodus Durone II______________________________________Control (no blanch) -- 87 97140° F. 30 116 114140° F. 60 114 146140° F. 120 128 164______________________________________ Normally, cherries are not blanched but processed in syrup. It is seen that low temperature blanch (140° F.) with 30, 60 and 120 minutes hold times gives increased firmness. EXAMPLE 8 Freestone peaches were cut in halves and blanched 20 minutes in water held at 145-150° F., held for 2 hours and then canned in 25 percent sugar syrup using a sterilization of 20 minutes at 212° F. Control peaches were canned with no blanch treatment which represents conventional commercial practice. Firmness was measured in a back extrusion cell. Four days later another lot of the same peaches were blanched 30 minutes and canned immediately with no hold time before sterilization. The peaches blanched 20 minutes at 145° F. with a 2 hour hold gave a firmness (newton's force) of 221, while those blanched 30 minutes at 145° F. with no hold gave a firmness of 237. Both results exceeded the control firmness of 174N. EXAMPLE 9 Golden Delicious apples were blanched 30 minutes (9A) and 60 minutes (9B) at 145° F. in water, then peeled, sliced and canned in 20 percent sugar syrup with a sterilization of 20 minutes at 212° F. Control apples were canned with no blanch which is the conventional process. Firmness was measured in the back extrusion cell. The firmness in newton's force, 223 for sample 9A and 265 for 9B, greatly exceeded the control of 89. EXAMPLE 10 It is well known in the food processing industry that the addition of compounds such as salts of calcium or magnesium impart firmer texture to processed vegetables and fruits. Two vegetables (green beans and potatoes) and one fruit (peaches) were canned with and without 0.07 percent added calcium chloride after being subjected to a conventional commercial blanch or a blanch in the 140°-150° F. range as described in the prior art examples. Firmness results using a back extrusion cell are given in the following table: TABLE 5__________________________________________________________________________ Blanch Hold Time FirmnessProduct Temperature (minutes) Without Calcium With Calcium__________________________________________________________________________Green beans 205° F. -- 218 266(BBL-47) 145° F. 30 476 646Potatoes no blanch (control) 378 362(white) 145° F. 15 405 402 145° F. 30 431 407Peaches no blanch (control) 174 221(Freestone) 20 min. @ 145° F. 120 221 269 30 min. @ 145° F. 0 237 289__________________________________________________________________________ The results in Table 5 above, show that a firmer product is produced in both green beans and peaches using either a 145° F. or a calcium treatment. A synergistic effect is noted for the combined treatment using both 145° F. blanch and calcium salt. Note the firmness of green beans increases to 646 with calcium and blanch versus 476 (blanch alone) as compared with 266 (calcium only) and 218 (no calcium), for the 205° F. blanch control. These limited tests showed no synergism for potatoes. EXAMPLE 11 It is known in the food industry that the heating regime needed to sterilize foods with a pH below about 4.5 is much less rigorous than for foods with a pH above 4.5 because heat resistant bacterial spores cannot grow below pH 4.5. This example is to show the effect of the combination of a milder heat sterilization with the addition of sufficient acid to bring the pH of a food having a pH higher than 4.5 to a pH of below 4.5. Carrots, green beans and cauliflower were given a conventional commercial blanch treatment, or a blanch in the 145°-150° range before processing with, and without, calcium and sufficient acid to reduce the pH below 4.5. The products without the addition of acid were given a commercial high temperature sterilization. The products with added acid were sterilized for 20 minutes at 212° F. which is sufficient to obtain commercial sterility because the pH was below 4.5. The results are shown in Table 6. Dramatic synergism is noted for carrots, green beans and cauliflower. For carrots an 8 minute blanch at 212° F. followed by sterilization using both calcium and citric acid gives a firmness of 2168 newton's force. In contrast, a blanch at 150° F. for 15 or 30 minutes using both calcium and citric acid gives firmness values of 3058 and 3485 newton's force. The combination of calcium plus citric acid and 145° F. blanch and 20 min. (212° F.) sterilization gives a 2168 firmness for cauliflower and 4376 for green beans. TABLE 6______________________________________Effect of Added Calcium and Acid on Firmness______________________________________ TreatmentProduct Blanch Sterilization______________________________________carrots, 8 min. at 212° F. 24 min. at 250° F., still retortDanvers sliced 8 min. at 212° F. -- 8 min. at 212° F. 20 min. at 212° F., still retort 15 min. at 150° F. 24 min. at 250° F., still retort -- -- -- 20 min. at 212° F., still retort 30 min. at 150° F. 24 min. at 250° F., still retort -- -- -- 20 min. at 212° F., still retortgreen beans 6 min. at 172° F. 8.5 min. at 250° F. inRBL-47 Steritort 4 min. at 145° F. -- (hold 30 min.) -- 20 min. at 212° F., still retortcauliflower 6 min. at 212° F. 22 min. at 240° F., still retort 10 min. at 145° F. -- (hold 30 min.) -- 20 min. at 212° F. in water______________________________________ TreatmentProduct Additives Firmness - Newtons Force______________________________________carrots, none 202Danvers sliced +Ca 279 +Ca + citric acid 2168 none 268 +Ca 362 +Ca + citric acid 3058 none 317 +Ca 440 +Ca + citric acid 3485green beans none 336RBL-47 none 454 +Ca + vinegar 4376cauliflower none 65 none 127 +Ca + citric acid 2168______________________________________ | Summary: Firmness of processed canned vegetables has been markedly improved by subjecting the vegetable to a low temperature blanching step at a temperature in the range of 125° F. to 160° F. and preferably from about 140° F. to 155° F. prior to conventional sterilization. Determination of blanch temperature conditions to produce optimum firmness in the processed food is made by first obtaining firmness values of specific foods at various blanch and hold temperatures and thereafter plotting the rate of firmness increase (Newtons/minute) against blanch temperatures. Preferred conditions, which vary for different foods, are obtained from individual plots for each food. Synergistic improvement of firmness results by combining the low temperature blanching with food grade acid (pH) and/or calcium salt additions. | 9,147 | 179 | big_patent | en |
Summarize: eh? The U.S.-Canadian Border Is Now.08 Percent Secure! Back in October, when Sharron Angle was regularly making unintentionally hilarious remarks on all manner of things, a minor diplomatic controversy erupted when she claimed that terrorists had entered America through the porous Canadian border. The Canadian ambassador to the United States took immediate action to rebut this slander on his country's good name. Her remarks led Canada's ambassador in Washington, Gary Doer, to write a letter to Angle's campaign on Monday. "There have been no terrorist attacks on the United States coming from Canada," he wrote, referencing the 9/11 Commission report. "I can assure you that Canada takes border security very seriously and trust you will see fit to set the record straight." As it turns out, though, Angle was, for once, correct. Angle never specified that she was referring to the 9/11 hijackers, just "the terrorists," and other would-be terrorists had indeed been caught crossing the Canadian border. Doer's letter continued: "We do not have a 'porous' border but rather one of the more secure borders in the world." Wrong again! Everyone already knew that security along the Canadian border was lacking, but a new government audit has now clarified just how abysmal it really is. "Only 32 miles of the nearly 4,000-mile U.S.-Canadian border have 'an acceptable level of security," the Buffalo News reports. Thirty-two miles! The News adds that, for security reasons, "the report did not specify which 32 miles of the U.S.-Canada border were considered secure and which were not." But if you're a terrorist, there's a pretty good chance that wherever you decide to cross is not one of the secure areas. About a 99.9 percent chance, actually. Report faults security along most of northern border [Buffalo News] WASHINGTON -- Only 32 miles of the nearly 4,000-mile U.S.-Canadian border have "an acceptable level of security," government auditors said Tuesday in a report that shows little progress almost a decade after the 9/11 terrorist attacks raised concerns about the porous nature of the nation's borders. The vast majority of northern border territory is "vulnerable to exploitation," the Government Accountability Office said in a report that lawmakers requested. The report was based on U.S. Border Patrol security assessments. Customs and Border Protection, which oversees the Border Patrol, "does not have the ability to detect illegal activity across most of the northern border," the government auditors said in the report. For national security reasons, the report did not specify which 32 miles of the U.S.-Canada border were considered secure and which were not during the 2010 federal fiscal year. But Rep. Brian Higgins, D-Buffalo, said he was worried that Buffalo's border is not as secure as it should be. The Peace Bridge does not meet Department of Homeland Security standards for modern, secure border crossings, he noted. For example, the Peace Bridge plaza lacks exit controls that could catch fugitives and does not have the space it needs for truck and bus inspections. The government audit comes less than a week after the Department of Homeland Security acknowledged it doesn't have the money to fund the reconstruction of the Peace Bridge plaza, which would fix those problems. "Given this demonstrated need, the Peace Bridge Expansion Project is something the federal government should make a top priority," Higgins said. Beyond Buffalo, the border's security problems are widespread, the report made clear. While the 9/11 terrorists did not cross into the U.S. from Canada, the Department of Homeland Security reports that the terrorist threat on the northern border is higher than on the Mexican border, auditors said, because of "the large expanse of area with limited law enforcement coverage." Given the lack of hands-on law enforcement in many border regions, auditors complained of "a high reliance on law enforcement support from outside the border zone." That lack of border law enforcement exists even though crime is common in U.S. and Canadian border regions. "DHS reports networks of illicit criminal activity and smuggling of drugs, currency, people and weapons between the two countries," the report said. Federal agents spent almost $3 billion to patrol the northern border in fiscal 2010 and made about 6,000 arrests, confiscating nearly 40,000 pounds of illegal drugs at the border. Nevertheless, auditors stressed that the federal government could do a much better job. Coordination among agencies -- a key post-9/11 goal and the reason for the merger of several agencies into the Department of Homeland Security -- has improved, auditors said. But there are still problems in key areas. For example, auditors noted that Immigration and Customs Enforcement officials "said they are reluctant to share intelligence information with Border Patrol because they are concerned Border Patrol may adversely affect an ICE investigation." One of the lawmakers who requested the report, Sen. Susan Collins, R-Maine, and Sen. Joe Lieberman of Connecticut, an independent, were outraged by its findings. "It is very clear from this report that the United States remains very vulnerable," Collins said. "It is truly shocking that we have total control of 32 miles of a 4,000-mile border." Lieberman said, "To me, this report is absolutely alarming." In a written response to the audit, the Department of Homeland Security agreed with the auditors' recommendation that the agencies within the department should better coordinate their security efforts at the northern border. North of the border, Canadian Immigration Minister Jason Kenney said Canada has improved border security, adding that there is no need for additional controls that would impede trade or travel, the Canadian Press reported. American lawmakers, however, were concerned. "We have an urgent need for smarter security investments that protect our northern border and our local economy," said Sen. Kirsten E. Gillibrand, D-N.Y. "It's clear we must do more to keep our border communities safe from suspected terrorists and drug trafficking." Sen. Charles E. Schumer, D-N.Y., who worked to create a narcotics strategy for the border, said: "In the coming weeks and months, I hope that Democrats and Republicans can come together to support innovative, high-tech solutions that will enhance border security." jzremski@buffnews.comnull | Summary: Nobody tell the terrorists: The US shares a 4,000-mile border with Canada, and 3,968 of those miles don't meet an "acceptable level" of security, says a new federal audit. Border Patrol "does not have the ability to detect illegal activity across most of the northern border," say the auditors, according to the Buffalo News. The report doesn't specify which 32 miles are safe, notes the News. "But if you're a terrorist, there's a pretty good chance that wherever you decide to cross is not one of the secure areas," adds New York magazine's Daily Intel. "About a 99.9% chance, actually." | 1,442 | 156 | multi_news | en |
Summarize: By. Daniel Bates and Daily Mail Reporter. PUBLISHED:. 19:24 EST, 8 January 2013. |. UPDATED:. 07:38 EST, 9 January 2013. The wife of a $1 million Chicago lottery winner who was poisoned with cyanide before he collected his winnings has revealed her husband was a 'good person' who didn't have any enemies. Shabana Ansari has opened up about losing 46-year-old Urooj Khan, who immigrated to the U.S. with from India in 1989. Describing Mr Khan as a hard-working, generous man who would send money to orphanages in their native country, Ms Ansari said her husband's death devastated her and added that she cannot fathom that he had any adversaries. 'I was shattered. I can't believe he's no longer with me,' she said, from one of the couple's three dry cleaners. SCROLL DOWN FOR VIDEO. Foul play: A family member called police to say Khan had not suffered a 'natural death' after he had been buried. His body will now be exhumed to inspect the contents of his last meal. Fateful purchase: Khan spent $60 in June on two Illinois state lottery tickets. The second one of which proved to be a winning ticket. Habit: Khan was revealed to have spent up to $600 a day on scratchcards. 'I don't think anyone would have a bad eye for him or that he had any enemy,' Ms Ansari said, adding that she continues to work at the dry cleaner out of a desire to honor her husband and the businesses he built. Ms Ansari, 32, moved to the U.S. from India after marrying Khan 12 years ago. Mr Khan and his wife were born in Hyderabad, India, and their story is a typical immigrant's tale of settling in a new land with big dreams and starting a business. Their daughter, Jasmeen, is a student here. 'Work was his passion,' Ms Ansari said. 'I'm just taking care of his hard work.' Ms Ansari wouldn't talk about the mysterious circumstances of her husband's death, saying it was too painful. But she says she hopes the truth will come out in the homicide investigation. Mr Khan's death on July 20 was initially ruled a result of natural causes. A relative's request for a deeper. look resulted in the conclusion months later that the father-of-one was. poisoned as he was about to collect $425,000 in winnings. The relative, who called a medical. examiner demanding a fresh inquiry into the poisoning, 'didn't accept it was going to be a natural. death,' MailOnline revealed earlier today. In a dramatic phone call, Mr Khan's. family member flatly rejected the original ruling and ordered. investigators to have another look. Entrepreneur: Khan emigrated to the U.S. during the 1980s, and had saved enough to open three dry cleaning shops (pictured) on Chicago's Far North side. Now. after further tests revealed Mr Khan, of Chicago's North Side, died due. to cyanide poisoning his body is set to be exhumed - so his final meal. can be examined. Chicago police Superintendent Garry McCarthy told reporters on Tuesday that he had never seen anything like Mr Khan's case in his 32 years of policing in New York, New Jersey and now Chicago. 'So, I'm not going to say that I've seen everything,' Mr McCarthy said. Prosecutors, Chicago police and the Cook County Medical Examiner's Office are investigating Khan's death as a homicide, but they have not given any details or announced any suspects. MailOnline. has also revealed that according to a respected poison expert, Mr Khan. would have ingested the killer dose no more than an hour before he died. According. to a police report cited by the Chicago Tribune, that fateful day Mr. Khan arrived at his Chicago home with his wife, Ms Ansari, and daughter,. Jasmeen, and ate some dinner. He then went to bed before waking up screaming as his body was'suffocated cell by cell.' Mr Khan won $1m on the Illinois State Lottery last June but went to collect his winnings on July 20. The. initial examination by the Cook County Medical examiner found he had. died of heart disease, or natural causes. Drugs like cyanide do not show. up on standard toxicology reports. However, after the intervention of a relative, a more thorough toxicology report was ordered. The cause of death has now been ruled as poisoning by cyanide and it is being treated as a homicide by Chicago police. The day after receiving a check for his winnings, Khan awoke screaming in pain in his bed at his Chicago home (pictured). An expert said this is caused when muscle contract suddenly as the poison kills you 'cell by cell' No signs of foul play: An initial autopsy on Khan performed by the Cook County Medical Examiner's Office found nothing awry. Cook County Medical Examiner Stephen. Cina told MailOnline: 'I believe they called up and said they wanted to. us to look into it further. 'They didn’t accept it was going to be a natural death.' Mr. Cina added that he hopes to exhume Mr Khan's body within ‘the next few. weeks’ from Chicago’s Rosehill Cemetery and that an analysis of the. content of his stomach could take place afterwards. Mr. Cina said: 'I have had one cyanide death in 4,500 autopsies so they are. unusual, as are exhumations. To have a case where there is both is very. unusual.' Mr Khan emigrated to the US during the. 1980s from India, and through hard work and discipline, saved up enough. cash to open first one and eventually three dry cleaning shops in. Chicago. Last June he paid. $60 for two lottery scratch-off tickets at a 7-Eleven convenience store near. his home and, upon scratching the second one, reportedly leaped in the. air and shouted, 'I hit a million!' Days. later, with his wife and daughter at his side at the same store, Mr. Khan gleefully accepted an oversized, mock check from lottery officials. At. the time, he said he was going to use the money to pay bills, donate to. St. Jude's Children's Hospital in Chicago and grow his dry-cleaning. business. After taxes, the prize money amounted to $425,000. Illinois State Lottery spokesman Mike Lang said that it was eventually cashed August 15 and that if a lottery winner dies, the money typically goes to his or her estate. He had recently returned to Chicago from the hajj pilgrimage to Saudi Arabia inspired to lead a better life and had sworn off buying lottery tickets - except just this once. His Islamic faith dictates that he should not even have been gambling in the first place. A good man: The cashier at the 7-Eleven where Khan bought his fateful lotto tickets said Khan was a 'gentle' man and a 'nice person' who cared for his family. Jimmy Goreel, 51, the manager of the. 7-Eleven store where Mr Khan bought the ticket, said that in 2011 Mr Khan. became ‘addicted’ to scratchcards and used to come in every day spending. up to $600 a time. He said:. 'Mr Khan would buy an entire book of scratchcards and would leave them. with me the next morning and ask me to work out how much he had won. 'That lasted about two months then he realised he had to slow down. 'He started getting addicted to it and he said he had to stop as he was a working person and a family man. A lot of the times he would come in his wife would come together with him.' Deborah. Blum, a poison expert whose book 'The Poisoner's Handbook' is being. made into a PBS TV series, said that Mr Khan would have been in. 'absolute agony' and likely coughed up blood as he was killed. A. former chemist, Miss Blum said: 'A good lethal dose of cyanide will. kill you in 10 minutes. A mid range dose and people die within the hour. For a poison that's pretty fast. 'Cyanide. poisoning is a nasty death. There is an enzyme which allows your cells. to breathe and the cyanide wipes that enzyme out. It suffocates you cell by cell. Symptoms include seizures, extreme shortness of breath and usually cardiac arrest. 'There. is something known as a 'cyanide scream' where your muscles are. contracting, so this could be what Mr Khan’s relatives heard.' Miss Blum added that cyanide would have had a bitter taste and that the best way to disguise it would be to put it into spicy food. A mere 200mg would be enough to take somebody's life, and that could be obtained from pesticides or certain kinds of rat poison. Historically, money is a 'good reason' why people poison, Miss Blum explained. She said: 'In the 19th century they used to call cyanide 'inheritance powder' because so many people used it to get their inheritance early. 'People also poison to punish, such as getting revenge on their boss, or to end a relationship that they want out of. 'Poisoners are unique killers because they are never impulsive. Poisoners are planners. 'There's always an element of calculation, and they think they will get away with it - just as nearly happened in this case.' Ms Ansari earlier told The Chicago Tribune: 'By God's grace, he was a workaholic. Day or night... he picks up the phone 24/7. He made the clients happy by doing his job. He could not be everywhere, but he had to be everywhere.' Chicago Police Department spokeswoman Melissa Stratton confirmed the department was now investigating the death. No arrests have so far been made | Summary: Shabana Ansari says Urooj Khan's July 20 death devastated her and that talking about how he died was too painful. Medical Examiner reveals family member called to say death of Mr Khan, 46, was foul play. Now preparing to exhume body and inspect contents of last meal. Poison expert said death would have been within hour and 'nasty' Worker at 7-Eleven where Mr Khan bought winning ticket reveals he would spent $600 a time on scratchcards. | 2,385 | 109 | cnn_dailymail | en |
Summarize: British woman dies after Everest trek October is peak trekking season in Nepal A British woman has died after being taken ill while on a trekking trip in Nepal's Everest region, police say. The 49-year-old was reportedly found dead in her hotel room after complaining of breathing difficulties. Local police told the AFP news agency she had just returned from a trek to Everest base camp, at 5,364m (17,700ft) above sea level, with her daughter. The Foreign Office said it was notified of the death and was providing consular assistance to the family. Police spokesman Purushottam Silwal said the pair were on their way to Nepal's capital Kathmandu when the woman experienced trouble breathing and decided to stop for a night at a hotel in Khumjung. He suggested the death could be down to altitude sickness, which leads to headaches, fatigue and dizziness and occurs when people ascend heights too quickly. The woman's body has been airlifted to Kathmandu for a post-mortem examination, he added. October is peak trekking season in Nepal, which is home to eight of the world's 14 highest mountains. AFP / Robert Kay A British woman died after returning from a trip to Everest base camp with her daughter A British woman on a trekking trip in Nepal's Everest region was found dead in her hotel room after complaining of respiratory problems, local police said Monday. The 49-year-old and her daughter were returning from a trip to Everest base camp, located at 5,364 metres (17,700 feet), when she experienced trouble breathing and stopped for a night at a hotel in Khumjung, en route to the capital Kathmandu. "She possibly died of altitude sickness," local police official, Purushottam Silwal, told AFP. "Her body was airlifted to Kathmandu for a post-mortem," Silwal said. Altitude sickness strikes when people ascend heights too quickly, as the decreased atmosphere pressure causes headaches, fatigue and dizziness. The incident follows the death of a Japanese climber last month, who slipped and fell during an ascent of Mount Manaslu, the world's eighth-highest peak. October is peak trekking season in the Himalayan nation, which is home to eight of the world's 14 highest mountains. Hundreds of climbers abandoned plans to ascend Mount Everest in April this year after 16 Nepalese guides were killed in an avalanche, marking the deadliest day on the 8,848-metre (29,029-foot) peak. | Summary: After the worst-ever Mount Everest disaster killed 16 Sherpa guides earlier this year, climbing season was cut short-but even getting to base camp can be deadly. A 49-year-old British woman who was trekking in the region died in her hotel room after returning from the camp, which is 17,700 feet above sea level, the BBC reports. The woman, who was trekking with her daughter, had complained of breathing difficulties. "She possibly died of altitude sickness," a local police official tells AFP. "Her body was airlifted to Kathmandu for a post-mortem." | 568 | 134 | multi_news | en |
Summarize: By. Associated Press. and Daily Mail Reporter. Police say a two-year-old boy playing in the front yard of an Indiana home with his seven-year-old sister may have been fatally struck by a bullet from a gunfight between two rival gangs about a block away. Officers suspect an argument began between the two groups around 6pm Wednesday a half-mile south of the University of Notre Dame, about a block from where two-year-old John Swoveland, Jr was playing in South Bend. One group is believed to have run west toward the boy and the other group continued shooting at them. Innocent victim: John Swoveland, Jr died after he was hit in the chest by a stray bullet while playing in front of his aunt's home. Family visit: The toddler was with his father and sister at his aunt's house when the shooting occurred. No arrests have been made and no suspects are in custody. Police are asking anyone with information to come forward. 'I'm tired of coming to see kids dead in the street,' Lieutenant David Wells told Fox 28. 'I'm just tired of it, so people need to band together and get with the program and start calling us, calling us at Homicide, with information. You don't even have to leave your name. I'm not even going to ask your name.' According to Fox 28, Metro Homicide officers believe two rival gangs exchanged gunfire near Arthur and Campeau Steets. One group ran west to escape the. other, and a stray bullet hit and killed the toddler in the front yard. of his aunt's home where he was playing. Police. also discovered a second shooting scene near Arthur and Campeau Streets. a few blocks east of Eddy Street, where there was evidence of gunfire,. but no one was known to be injured. Devastated family: The relatives of the toddler are in disbelief over the two-year-old's death. Related: Police believe the death of John Swoveland, Jr is linked to another shooting incident just blocks away. The little boy's mother has been named as Ashley Kinder, 23. She is currently expecting another child. Before John Swoveland, Jr's birth Kinder suffered another tragedy when her baby daughter died of natural causes at just nine days old. The boy's father John Swoveland was in the house at the time of the shooting. 'I didn't see nothing. I was, just went inside to get, to get my girlfriend's attention for her to come outside and play with us and I, my hand on the door still my little girl comes inside and says someone just drove by and shot my son, and I walked outside and my son's all white and he's just laying on the ground all messed up,' Swoveland told Fox28. Prosecutors say the autopsy on John Swoveland, Jr. Is complete. The forensic pathologist found the cause of death to be a gunshot wound to the chest and the manner of death to be homicide | Summary: Two-year-old John Swoveland, Jr was playing with his sister, seven, in their aunt's front yard Wednesday at 6pm. The toddler was hit in the chest by a bullet and died. Police believe the bullet may have come from a gunfight between rival gangs a block away. No suspects have been named and no arrests have been made. The little boy's mother Ashley Kinder, 23, is heavily pregnant. She previously lost a baby girl who died of natural causes at nine days old. Police are asking anyone with information to come forward. | 686 | 125 | cnn_dailymail | en |
Write a title and summarize: SECTION 1. ELIMINATION OF ANNUAL CAP ON AMOUNT OF MEDICARE PAYMENT FOR OUTPATIENT PHYSICAL THERAPY AND OCCUPATIONAL THERAPY SERVICES. (a) In General.--Section 1833 of the Social Security Act (42 U.S.C. 1395l) is amended by striking subsection (g). (b) Effective Date.--The amendment made by subsection (a) shall apply to services furnished on or after January 1, 1994. SEC. 2. EXTRA-BILLING LIMITS. (a) Enforcement and Uniform Application.-- (1) Enforcement.--Paragraph (1) of section 1848(g) of the Social Security Act (42 U.S.C. 1395w-4(g)) is amended to read as follows: ``(1) Limitation on actual charges.-- ``(A) In general.--In the case of a nonparticipating physician or nonparticipating supplier or other person (as defined in section 1842(i)(2)) who does not accept payment on an assignment-related basis with respect to a physician's service furnished to an individual enrolled under this part, the following rules apply: ``(i) Application of limiting charge.--No such physician, supplier, or person may bill or collect an actual charge for the service in excess of the limiting charge described in paragraph (2) for such service. ``(ii) No liability for excess charges.--No person is liable for payment of any amounts billed for the service in excess of such limiting charge. ``(iii) Correction of excess charges.--If such a physician, supplier, or other person bills, but does not collect, an actual charge for a service in violation of clause (i), the physician, supplier, or other person shall reduce on a timely basis the actual charge billed for the service to an amount not to exceed the limiting charge for the service. ``(iv) Refund of excess collections.--If such a physician, supplier, or other person collects an actual charge for a service in violation of clause (i), the physician, supplier, or other person shall provide on a timely basis a refund to the individual charged in the amount by which the amount collected exceeded the limiting charge for the service. The amount of such a refund shall be reduced to the extent the individual has an outstanding balance owed by the individual to the physician, supplier, or other person. ``(B) Sanctions.--If a physician, supplier, or other person-- ``(i) knowingly and willfully bills or collects for services in violation of subparagraph (A)(i) on a repeated basis, or ``(ii) fails to comply with clause (iii) or (iv) of subparagraph (A) on a timely basis, the Secretary may apply sanctions against the physician, supplier, or other person in accordance with paragraph (2) of section 1842(j). The provisions of section 1842(j)(4) shall apply for purposes of this paragraph except that any reference in such section to a physician is deemed also to include a reference to a supplier or other person under this subparagraph. ``(C) Timely basis.--For purposes of this paragraph, the term `on a timely basis', means not later than 30 days after the date the physician, supplier, or other person is notified by the carrier under this part of a violation of the requirements of subparagraph (A).''. (2) Uniform application of extra-billing limits to physicians' services.-- (A) In general.--Section 1848(g)(2)(C) of the Social Security Act (42 U.S.C. 1395w-4(g)(2)(C)) is amended by inserting ``or for nonparticipating suppliers or other persons'' after ``nonparticipating physicians''. (B) Conforming definition.--Section 1842(i)(2) of the Social Security Act (42 U.S.C. 1395u(i)(2)) is amended-- (i) by striking ``, and the term'' and inserting ``; the term'', and (ii) by inserting before the period at the end the following: ``; and the term `nonparticipating supplier or other person' means a supplier or other person (excluding a provider of services) that is not a participating physician or supplier (as defined in subsection (h)(1))''. (3) Additional conforming amendments.--Section 1848 of the Social Security Act (42 U.S.C. 1395w-4) is amended-- (A) in subsection (a)(3)-- (i) by inserting ``and suppliers'' after ``physicians'', (ii) by inserting ``or a nonparticipating supplier or other person (as defined in section 1842(i)(2))'' after ``nonparticipating physician'', and (iii) by adding at the end the following: ``In the case of physicians' services (including services which the Secretary excludes pursuant to subsection (j)(3)) of a nonparticipating physician, supplier, or other person for which payment is made under this part on a basis other than the fee schedule amount, the payment shall be based on 95 percent of the payment basis for services of such type which are furnished by a participating physician, supplier, or other person.''; (B) in subsection (g)(1)(A), as amended by subsection (a), in the matter before clause (i), by inserting ``(including services which the Secretary excludes pursuant to subsection (j)(3))'' after ``a physician's service''; (C) in subsection (g)(2)(D), by inserting ``(or, if payment under this part is made on a basis other than the fee schedule under this section, 95 percent of the other payment basis)'' after ``subsection (a)''; (D) in subsection (g)(3)(B)-- (i) by inserting after the first sentence the following: ``No person is liable for payment of any amounts billed for such a service in violation of the previous sentence.'', and (ii) in the last sentence, by striking ``previous sentence'' and inserting ``first sentence''; and (E) in subsection (h)-- (i) by inserting ``or nonparticipating supplier or other person'' after ``physician'' the first place it appears, (ii) by inserting ``, supplier, or other person'' after ``physician'' the second place it appears, and (iii) by inserting ``, suppliers, and other persons'' after ``physicians'' the second place it appears. (b) Information on Extra-Billing Limits.-- (1) Part of explanation of medicare benefits.--Section 1842(h)(7) of the Social Security Act (42 U.S.C. 1395u(h)(7)) is amended-- (A) by striking ``and'' at the end of subparagraph (B); (B) in subparagraph (C), by striking ``shall include'' and by striking the period at the end and inserting ``, and''; and (C) by adding at the end the following new subparagraph: ``(D) in the case of services for which the billed amount exceeds the limiting charge imposed under section 1848(g), information regarding such limiting charge (including information concerning the right to a refund under section 1848(g)(1)(A)(iv)).''. (2) Determinations by carriers.--Subparagraph (G) of section 1842(b)(3) of the Social Security Act (42 U.S.C. 1395u(b)(3)) is amended to read as follows: ``(G) for a service that is furnished with respect to an individual enrolled under this part, that is not paid on an assignment-related basis, and that is subject to a limiting charge under section 1848(g), will-- ``(i) determine, prior to making payment, whether the amount billed for such service exceeds the limiting charge applicable under section 1848(g)(2); ``(ii) notify the physician, supplier, or other person periodically (but not less often than once every 30 days) of determinations that amounts billed exceeded such limiting charges; and ``(iii) provide for prompt response to inquiries of physicians, suppliers, and other persons concerning the accuracy of such limiting charges for their services;''. (c) Report on Charges in Excess of Limiting Charge.--Section 1848(g)(6)(B) of the Social Security Act (42 U.S.C. 1395w-4(g)(6)(B)) is amended by inserting ``on the extent to which actual charges exceed limiting charges, the number and types of services involved, and the average amount of excess charges and'' after ``report to the Congress''. (d) Effective Dates.-- (1) Enforcement and uniform application.--The amendments made by subsection (a) shall apply to services furnished on or after January 1, 1994. (2) Explanations.--The amendments made by subsection (b)(1) shall apply to explanations of benefits provided on or after January 1, 1994, except that the requirement for including information concerning the right to a refund shall apply to explanations of benefits provided on or after July 1, 1994. (3) Carrier determinations.--The amendments made by subsection (b)(2) shall apply to contracts as of January 1, 1994. (4) Report.--The amendment made by subsection (c) shall apply to reports for years beginning after 1994. | Title: To amend title XVIII of the Social Security Act to eliminate the annual cap on the amount of payment for outpatient physical therapy and occupational therapy services under part B of the medicare program, and for other purposes Summary: Amends title XVIII (Medicare) of the Social Security Act to eliminate the annual cap on the amount of payment for outpatient physical therapy and occupational therapy services under Medicare part B (Supplementary Medical Insurance). Revises the limitation on beneficiary liability for payment of any amounts billed in excess of the applicable limiting charge for physician services. Applies such limitation to nonparticipating suppliers and other persons, as well as nonparticipating physicians. Includes in the Secretary of Health and Human Services' annual explanation of Medicare benefits information on refunds of such amounts. Makes carriers responsible for determining, prior to making payment, whether the amount billed for services is in excess of the applicable limiting charge and, if so, notifying the physician or other providers as appropriate. Requires the reports to the Congress on changes in excess charges for physician services to reflect additional information on the services involved. | 2,328 | 229 | billsum | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``National Defense Enhancement and National Guard Empowerment Act of 2006''. SEC. 2. EXPANDED AUTHORITY OF CHIEF OF THE NATIONAL GUARD BUREAU AND EXPANDED FUNCTIONS OF THE NATIONAL GUARD BUREAU. (a) Expanded Authority.-- (1) In general.--Subsection (a) of section 10501 of title 10, United States Code, is amended by striking ``joint bureau of the Department of the Army and the Department of the Air Force'' and inserting ``joint activity of the Department of Defense''. (2) Purpose.--Subsection (b) of such section is amended by striking ``between'' and all that follows and inserting ``between-- ``(1)(A) the Secretary of Defense, the Joint Chiefs of Staff, and the commanders of the combatant commands for the United States, and (B) the Department of the Army and the Department of the Air Force; and ``(2) the several States.''. (b) Enhancements of Position of Chief of the National Guard Bureau.-- (1) Advisory function on national guard matters.-- Subsection (c) of section 10502 of title 10, United States Code, is amended by inserting ``to the Secretary of Defense, to the Chairman of the Joint Chiefs of Staff,'' after ``principal advisor''. (2) Member of joint chiefs of staff.--(A) Such section is further amended-- (i) by redesignating subsection (d) as subsection (e); and (ii) by inserting after subsection (c) the following new subsection (d): ``(d) Member of Joint Chiefs of Staff.--The Chief of the National Guard Bureau shall perform the duties prescribed for him or her as a member of the Joint Chiefs of Staff under section 151 of this title.''. (B) Section 151(a) of such title is amended by adding at the end the following new paragraph: ``(7) The Chief of the National Guard Bureau.''. (3) Grade.--Subsection (e) of such section, as redesignated by paragraph (2)(A)(i) of this subsection, is further amended by striking ``lieutenant general'' and inserting ``general''. (4) Annual report to congress on validated requirements.-- Section 10504 of such title is amended by adding at the end the following new subsection: ``(c) Annual Report on Validated Requirements.--Not later than December 31 each year, the Chief of the National Guard Bureau shall submit to Congress a report on the following: ``(1) The requirements validated under section 10503a(b)(1) of this title during the preceding fiscal year. ``(2) The requirements referred to in paragraph (1) for which funding is to be requested in the next budget for a fiscal year under section 10544 of this title. ``(3) The requirements referred to in paragraph (1) for which funding will not be requested in the next budget for a fiscal year under section 10544 of this title.''. (c) Enhancement of Functions of National Guard Bureau.-- (1) Development of charter.--Section 10503 of title 10, United States Code, is amended-- (A) in the matter preceding paragraph (1), by striking ``The Secretary of the Army and the Secretary of the Air Force shall jointly develop'' and inserting ``The Secretary of Defense, in consultation with the Secretary of the Army and the Secretary of the Air Force, shall develop''; and (B) in paragraph (12), by striking ``the Secretaries'' and inserting ``the Secretary of Defense''. (2) Additional general functions.--Such section is further amended-- (A) by redesignating paragraph (12), as amended by paragraph (1)(B) of this subsection, as paragraph (13); and (B) by inserting after paragraph (11) the following new paragraph (12): ``(12) Facilitating and coordinating with other Federal agencies, and with the several States, the use of National Guard personnel and resources for and in contingency operations, military operations other than war, natural disasters, support of civil authorities, and other circumstances.''. (3) Military assistance for civil authorities.--Chapter 1011 of such title is further amended by inserting after section 10503 the following new section: ``Sec. 10503a. Functions of National Guard Bureau: military assistance to civil authorities ``(a) Identification of Additional Necessary Assistance.--The Chief of the National Guard Bureau shall-- ``(1) identify gaps between Federal and State capabilities to prepare for and respond to emergencies; and ``(2) make recommendations to the Secretary of Defense on programs and activities of the National Guard for military assistance to civil authorities to address such gaps. ``(b) Scope of Responsibilities.--In meeting the requirements of subsection (a), the Chief of the National Guard Bureau shall, in coordination with the Adjutant Generals of the States, have responsibilities as follows: ``(1) To validate the requirements of the several States and Territories with respect to military assistance to civil authorities. ``(2) To develop doctrine and training requirements relating to the provision of military assistance to civil authorities. ``(3) To acquire equipment, materiel, and other supplies and services for the provision of military assistance to civil authorities. ``(4) To assist the Secretary of Defense in preparing the budget required under section 10544 of this title. ``(5) To administer amounts provided the National Guard for the provision of military assistance to civil authorities. ``(6) To carry out any other responsibility relating to the provision of military assistance to civil authorities as the Secretary of Defense shall specify. ``(c) Assistance.--The Chairman of the Joint Chiefs of Staff shall assist the Chief of the National Guard Bureau in carrying out activities under this section. ``(d) Consultation.--The Chief of the National Guard Bureau shall carry out activities under this section in consultation with the Secretary of the Army and the Secretary of the Air Force.''. (4) Budgeting for training and equipment for military assistance to civil authorities and other domestic missions.-- Chapter 1013 of title 10, United States Code, is amended by adding at the end the following new section: ``Sec. 10544. National Guard training and equipment: budget for military assistance to civil authorities and for other domestic operations ``(a) In General.--The budget justification documents materials submitted to Congress in support of the budget of the President for a fiscal year (as submitted with the budget of the President under section 1105(a) of title 31) shall specify separate amounts for training and equipment for the National Guard for purposes of military assistance to civil authorities and for other domestic operations during such fiscal year. ``(b) Scope of Funding.--The amounts specified under subsection (a) for a fiscal year shall be sufficient for purposes as follows: ``(1) The development and implementation of doctrine and training requirements applicable to the assistance and operations described in subsection (a) for such fiscal year. ``(2) The acquisition of equipment, materiel, and other supplies and services necessary for the provision of such assistance and such operations in such fiscal year.''. (5) Limitation on increase in personnel of national guard bureau.--The Secretary of Defense shall, to the extent practicable, ensure that no additional personnel are assigned to the National Guard Bureau in order to address administrative or other requirements arising out of the amendments made by this subsection. (d) Conforming and Clerical Amendments.-- (1) Conforming amendment.--The heading of section 10503 of such title is amended to read as follows: ``Sec. 10503. Functions of National Guard Bureau: charter''. (2) Clerical amendments.--(A) The table of sections at the beginning of chapter 1011 of such title is amended by striking the item relating to section 10503 and inserting the following new items: ``10503. Functions of National Guard Bureau: charter. ``10503a. Functions of National Guard Bureau: military assistance to civil authorities.''. (B) The table of sections at the beginning of chapter 1013 of such title is amended by adding at the end the following new item: ``10544. National Guard training and equipment: budget for military assistance to civil authorities and for other domestic operations.''. (e) Termination of Position of Assistant to Chairman of Joint Chiefs of Staff for National Guard Matters.--Section 901 of the National Defense Authorization Act for Fiscal Year 1998 (Public Law 105-85; 111 Stat. 1853; 10 U.S.C. 155 note) is amended to read as follows: ``SEC. 901. ASSISTANT TO THE CHAIRMAN OF THE JOINT CHIEFS OF STAFF FOR RESERVE MATTERS. ``(a) In General.--There is within the Joint Staff the position of Assistant to the Chairman of the Joint Chiefs of Staff for Reserve Matters. ``(b) Selection.--The Assistant to the Chairman of the Joint Chiefs of Staff for Reserve Matters shall be selected by the Chairman from officers of the Army Reserve, the Navy Reserve, the Marine Corps Reserve, or the Air Force Reserve who-- ``(1) are recommended for such selection by the Secretary of the military department concerned; ``(2) have had at least 10 years of commissioned service in their reserve component; and ``(3) are in a grade above colonel or, in the case the Navy Reserve, captain. ``(c) Term of Office.--The Assistant to the Chairman of the Joint Chiefs of Staff for Reserve Matters serves at the pleasure of the Chairman for a term of two years and may be continued in that assignment in the same manner, for one additional term. However, in a time of war there is no limit on the number of terms. ``(d) Grade.--The Assistant to the Chairman of the Joint Chiefs of Staff for Reserve Matters while so serving, holds the grade of major general or, in the case of the Navy Reserve, rear admiral. The officer serving in the position shall be considered to be serving in a position external to that officer's Armed Force for purposes of section 721 of title 10, United States Code. ``(e) Duties.--The Assistant to the Chairman of the Joint Chiefs of Staff for Reserve Matters is an advisor to the Chairman on matters relating to the reserves and performs the duties prescribed for the position by the Chairman. ``(f) Other Reserve Component Representation on Joint Staff.--The Secretary of Defense, in consultation with the Chairman of the Joint Chiefs of Staff, shall develop appropriate policy guidance to ensure that, to the maximum extent practicable, the level of reserve component officer representation within the Joint Staff is commensurate with the significant role of the reserve components within the Total Force.''. SEC. 3. PROMOTION OF ELIGIBLE RESERVE OFFICERS TO LIEUTENANT GENERAL AND VICE ADMIRAL GRADES ON THE ACTIVE-DUTY LIST. (a) Sense of Congress.--It is the sense of Congress that, whenever officers are considered for promotion to the grade of lieutenant general, or vice admiral in the case of the Navy, on the active duty list, officers of the reserve components of the Armed Forces who are eligible for promotion to such grade should be considered for promotion to such grade. (b) Proposal.--The Secretary of Defense shall submit to Congress a proposal for mechanisms to achieve the objective specified in subsection (a). The proposal shall include such recommendations for legislative or administrative action as the Secretary considers appropriate in order to achieve that objective. (c) Notice Accompanying Nominations.--The President shall include with each nomination of an officer to the grade of lieutenant general, or vice admiral in the case of the Navy, on the active-duty list that is submitted to the Senate for consideration a certification that all reserve officers who were eligible for consideration for promotion to such grade were considered in the making of such nomination. SEC. 4. REQUIREMENT THAT POSITION OF DEPUTY COMMANDER OF THE UNITED STATES NORTHERN COMMAND BE FILLED BY A QUALIFIED NATIONAL GUARD OFFICER. (a) In General.--The position of Deputy Commander of the United States Northern Command shall be filled by a qualified officer of the National Guard who is eligible for promotion to the grade of lieutenant general. (b) Purpose.--The purpose of the requirement in subsection (a) is to ensure that information received from the National Guard Bureau regarding the operation of the National Guard of the several States is integrated into the plans and operations of the United States Northern Command. | Title: To amend title 10, United States Code, to enhance the national defense through empowerment of the Chief of the National Guard Bureau and the enhancement of the functions of the National Guard Bureau, and for other purposes Summary: National Defense Enhancement and National Guard Empowerment Act of 2006 - Expands the: (1) authority of the Chief of the National Guard Bureau (Bureau) to include membership on the Joint Chiefs of Staff (JCS) (and raises the grade of the Chief from lieutenant general to general); and (2) functions of the Bureau to include facilitating and coordinating, with other federal agencies and the states, the use of Guard personnel and resources for, and in, contingency operations, military operations other than war, natural disasters, and support of civil authorities. Directs the Chief to: (1) identify gaps between federal and state capabilities to prepare for and respond to emergencies; and (2) make recommendations to the Secretary of Defense on Guard programs and activities to address such gaps. Requires annual Department of Defense (DOD) budget justification documents to include separate amounts for Guard training and equipment for military assistance to civil authorities and other domestic operations. Establishes within the JCS an Assistant to the Chairman of the Joint Chiefs of Staff for Reserve Matters. Expresses the sense of Congress calling for consideration of eligible reserve officers for promotion to the grades of lieutenant general or vice admiral on the active duty list. Requires the position of Deputy Commander of the U.S. Northern Command to be filled by a qualified Guard officer eligible for promotion to the grade of lieutenant general. | 2,990 | 343 | billsum | en |
Summarize: By. Leon Watson. PUBLISHED:. 08:32 EST, 27 February 2014. |. UPDATED:. 16:32 EST, 27 February 2014. A family have told how their son's deteriorating condition in hospital turned into a heart-breaking custody battle that stripped them of their right to visit their child. Bret Bohn, 27, went to hospital in his home state of Alaska after developing a relatively minor case of nasal polyps - overgrowths in the nose - last fall. He was an athletic, healthy man who worked as a field guide for hunters. The Bohn family are fighting to visit their son Bohn, who has been declared a ward of the state in Alaska. Bret Bohn was an athletic, healthy man who worked as a field guide for hunters in Alaska before he went to hospital. But after the polyps were surgically removed, the growths came back and he was prescribed Prednisone, an anti-inflammatory medication. Mr Bohn's family say it had a devastating effect on him. Soon afterwards, he started to have trouble sleeping, they said. In October, his parents, Glenn and Lorraine, took their son. to the Providence Medical Center in Anchorage for severe insomnia. Doctors there. prescribed drugs and sent him home. Mr Bohn's health then deteriorated rapidly and, after a. seizure, his family decided to take him back to the hospital, TheBlaze reported. In the hospital, Mr Bohn was unable to. sleep for some 24 days and his mental faculties were significantly. diminished. His parents said that at this point they assumed power of. attorney over him, using a written agreement allowing them to make. medical decisions for him. Mr Bohn's parents Lorraine and Glenn said they assumed power of attorney over their son, using a written agreement allowing them to make medical decisions for him. More than 35 lab tests were conducted to diagnose Mr Bohn at Providence Medical Center (pictured) in Anchorage. That agreement was initially drawn up in. 2007, when Mr Bohn was a healthy 20-year-old. More. than 35 lab tests were conducted to diagnose Mr Bohn. Meanwhile, his family said, doctors were medicating him with. dozens of drugs, rendering him in a state of 'delirium.' At one point,. they said, Mr Bohn became so frustrated that he attempted to leave the. hospital on his own, but was talked down by his parents. That's. when his family, who contend the hospital's course of treatment made. their son worse, asked for a second opinion or different course of. medical action. They say they were denied and were not permitted to. withdraw their son from the hospital. Eventually, a custody battle broke out. A judge ultimately ruled in favor of the state. Mr Bohn's mother Lorraine said: 'It's a nightmare that this even could be happening. I'm heartbroken, very heartbroken' Now, Mr Bohn is a ward of the. state and has been diagnosed with a mental-disorder, which has resulted. in doctors heavily medicating him with various drugs, his family said. 'I hurt — I cry every day and every night. It's a nightmare,' Mr Bohn's mother Lorraine told KTUU-TV. 'It's a nightmare that this even could be happening. I'm heartbroken, very heartbroken. 'You know, I can't help but to blame myself,' she said. The last time Mr Bohn's saw him was during a short supervised Christmas Day visit and his birthday was last month, according to The Blaze. Barbara Dick, of Alaska's Adult Protective Services department, said: 'We can't just come in and take away somebody's right and say, "That's it," 'We have to take it to court and we have our state attorneys with us and we have to have the evidence to support that.' A spokesman for Providence Medical Center said he was unable to comment to on the case due to privacy laws. But he did say: 'Health care providers are required by state law to make reports of harm to Adult Protective Services whenever they have reasonable cause to believe a vulnerable adult suffers from abuse or neglect. 'Health care providers are permitted under state law, and required by their standard of care, to decline to comply with the direction of a surrogate if they determine that the surrogate is not abiding by the wishes, values, and best interest of the patient.' A Facebook page called 'Free Bret Bohn,' shows individuals picketing, demanding the 27-year-old's release | Summary: Bret Bohn went to hospital after developing a relatively minor ailment. The 27-year-old had been an athletic, healthy field guide in Alaska. But his health then went downhill after he was proscribed drugs. Mr Bohn's parents tried to have him discharged - but were refused. Doctors at the hospital then assumed power of. attorney over him. Mr Bohn's family have been in a custody battle for him ever since. The last time they saw him was a supervised visit on Christmas Day. | 1,077 | 117 | cnn_dailymail | en |
Summarize: Background Doping in sport involves the use of substances or methods that artificially enhance athletic performance. Although doping is not a new phenomenon, policymakers remain concerned about the health effects and ethical implications associated with the continued use of performance-enhancing substances in competitive sports. Anti-doping rules have been in place for various sports and in various countries since at least the 1920s, but many claim that the enforcement of anti-doping rules is fundamentally limited by a lack of uniform standards across sports and across countries. The International Convention Against Doping in Sport, adopted in 2005 by UNESCO, is the most recent response to such criticism. It seeks to harmonize anti-doping commitments for sport. Eighty-seven states are parties, or States Parties, to the Convention as of July 2008. Treaty Evolution The field of international anti-doping policy has undergone several rapid transformations in the past decade. The International Olympic Committee (IOC), the umbrella organization for all participants in the Olympic Movement and whose authority includes sport doping regulation, leads the international campaign against sport doping. In 1999, the IOC created the World Anti-Doping Agency (WADA) as the first independent entity to monitor and enforce international anti-doping activities for non-professional sports. One of WADA's first achievements was to develop an international framework for harmonizing anti-doping policies, rules, and regulations among international amateur sport organizations and public authorities, called the World Anti-Doping Code (the Code). As a non-governmental foundation, however, WADA cannot legally bind governments to its policies, including the Code. In 2003, 191 countries, including the United States, signed the Copenhagen Declaration on Anti-Doping in Sport. The Copenhagen Declaration is a political statement of intent to identify the Code as the "foundation in the world wide fight against doping in sport," ensure that national anti-doping policies conform with the Code, and acknowledge the role of WADA in coordinating and standardizing international anti-doping efforts according to the Code. UNESCO's 2005 International Convention Against Doping in Sport further institutionalizes anti-doping norms among State Parties. Treaty Commitments States Parties to the Convention are required to harmonize national laws, regulations, policies, and administrative practices with the "principles" of the Code. This includes restricting availability of prohibited substances, applying WADA's standards for granting therapeutic use exemptions, funding domestic anti-doping test programs, supporting the sanctioning of doping violators, promoting anti-doping research and education, and facilitating international anti-doping cooperation by supporting and funding WADA. Every two years, States Parties are also required to provide the Conference of Parties with status reports on their compliance with the Convention. States Parties are the sole voting members of the Convention's Conference of Parties. WADA is an advisory organization to the Conference of Parties. U.S. ratification does not require changes to domestic laws nor additional funding contribution levels to WADA and domestic anti-doping programs. Further, the Convention does not apply to U.S. professional sport associations and institutions. U.S. Policy The Administration states that ratification of this treaty is a priority. On February 6, 2008, the President transmitted the treaty to the Senate for its advice and consent to ratification. Upon the President's transmittal of the Convention to the Senate for consideration, the White House Press Secretary released a statement justifying the need for ratification, stating that it would "solidify our Nation's place as a leader in the worldwide effort to rid athletics of cheating through chemistry." Many, including Members of Congress, argue that international commitments to anti-doping can help set a positive example for elite and aspiring athletes and help discourage their use of performance-enhancing substances. A major impetus for U.S. action on ratifying this treaty stems from a concern that lack of ratification would result in a ban from hosting and participating in some international competitions, including future Olympic Games. The IOC requires that governments become a party to the International Convention Against Doping in Sport by January 1, 2009—and failure to do so would result in those countries being barred from participation in future games and could also affect a city's bid for hosting the 2016 Olympics. Chicago is the U.S. Olympic Committee's candidate for the 2016 Games. The IOC plans to announce its selection for the 2016 Olympics in October 2009. Ratification Status On August 4, 2008, President Bush signed the instrument of ratification for the treaty. Prior to this date, the Senate Foreign Relations Committee held a hearing on the treaty on May 22, 2008. On June 24, 2008, the committee approved the treaty by voice vote, and the Senate provided its advice and consent to ratification on July 21, 2008 (Senate Congressional Record, Page S6980), subject to an understanding, a declaration, and a condition. The understanding states that the treaty does not obligate the U.S. government to provide funding to WADA. The declaration limits the definition of an athlete for the purposes of doping control to those that the U.S. Anti-Doping Agency (USADA) determines is subject to WADA. The condition states that the text of amendments to the treaty's annexes be transmitted to Congress within 60 days after an amendment occurs. National Anti-doping Testing Since its creation in October 2000, the U.S. Anti-Doping Agency (USADA) has implemented and enforced the Code for Olympic sports in the United States. In 2006, USADA reported spending $5.9 million on anti-doping test expenses, primarily for Olympic, Paralympic, and Pan American Sports. Professional sport associations and leagues, including Major League Baseball and the National Football League, do not fall under the jurisdiction of federal regulation and adhere to their own anti-doping policies, testing requirements, and penalties. Substance Control The U.S. Office on National Drug Control Policy, the lead policy office for national drug control programs, identifies the elimination of doping in sport as a goal in its 2008 National Drug Control Strategy document. To this end, several national laws proscribe many substances identified by the Code. The Controlled Substances Act, as amended (21 U.S.C. 801 et seq.), prohibits the production, movement, import, distribution, and sale of many of the substances identified in the Code's "List of Prohibited Substances and Methods." Substances on the Code's list that are not subject to the Controlled Substances Act are subject to the Federal Food, Drug, and Cosmetic Act, as amended (21 U.S.C. 301 et seq.), which restricts their use to legitimate medical activities. In addition, the U.S. government commits law enforcement resources to investigate doping cases. In one example, the U.S. Drug Enforcement Administration (DEA), in conjunction with other law enforcement entities, participated in a two-year international steroid trafficking investigation ending in 2007, called Operation Raw Deal, which resulted in 124 arrests and the seizure of 11.4 million steroid dosage units, $6.5 million, 25 vehicles, three boats, and 71 weapons. Commitments to Anti-doping Research and Education USADA and U.S. agencies support anti-doping research and education. USADA budgets approximately $2 million per year in support of research related to the deterrence of the use of performance-enhancing drugs in sports, and in 2006, spent approximately $1.3 million in education. Combined, anti-doping research and education programs accounted for approximately 27% of USADA's expenses in that year. Other anti-doping research is funded through the U.S. Department of Health and Human Services, DEA, and U.S. Department of Education. Multilateral Commitments The Administration demonstrates its support for international efforts to combat doping in sport through its participation in several multilateral anti-doping venues. As a signatory to the 2003 Copenhagen Declaration on Anti-Doping in Sport, the U.S. government formally recognized the role and importance of WADA in harmonizing international anti-doping standards. The U.S. government is also a member of WADA's Foundation Board and Executive Committee, and Congress regularly appropriates funding to support WADA. In FY2008, Congress appropriated $1.7 million to WADA. The United States also participates in anti-doping activities through the Council of Europe, the Caribbean Community, and the Americas Sports Council, the latter an informal governmental organization designed to combat doping in sport in North, South, and Central America. Issues for Consideration Consequences for Professional Sports The International Convention Against Doping in Sport does not apply to U.S. professional sports. Some athletes of U.S. professional teams, however, may already follow the Code—whose guidelines the Convention embraces—when they participate in international tournaments under the jurisdiction of organizations that already implement the Code. This has already been the case, for example, when National Basketball Association or National Hockey League players participate in the Olympic Games or World Championships. Article 20 of the Convention, however, requires that States Parties encourage professional sports associations and institutions to develop anti-doping principles consistent with the Code. Effectiveness of Anti-doping Tests Many observers argue that the value of international anti-doping efforts, including the International Convention Against Doping in Sport, are limited by a lack of effective anti-doping tests that can positively identify prohibited substances. Historically, the search for better methods to detect doping substances has lagged behind the discovery and use of new, undetectable substances. For example, though the use of anabolic steroids was already reportedly "widespread" by the early 1970s, a reliable test method for detecting them was not found until 1974, and they were only added to the IOC's list of prohibited substances in 1976. More recently, anti-doping efforts suffered setbacks related to the use of designer, or synthetic, steroids that have proven difficult to detect. New concern currently surrounds the possibility of "gene doping," whereby genetic therapies may be used to enhance athletic performance. Athletes' Rights Protection The International Convention Against Doping in Sport requires States Parties to adhere to the principles of the Code. Some observers, however, question whether the Code sufficiently protects athletes who may be accidentally implicated in a doping violation. These critics argue that the Code imposes excessively stringent punishments, including potentially career-ending suspensions, that offer little distinction between intentional doping and the detection of trace levels of prohibited substances originating from the consumption of contaminated or mislabeled nutritional supplements. Some of these concerns may be addressed in revised version of the Code, which will go into effect January 1, 2009. Further, some question the legitimacy of WADA as an authority in international anti-doping policy, accusing WADA of not being sufficiently transparent in its decision making and resistant to outside scrutiny. Some critics have also questioned WADA procedures for handling drug testing samples and avoiding contamination, as well as for dealing with false positives and other potential testing mistakes. Cost of Enforcing Anti-doping Regulations The International Convention Against Doping in Sport commits States Parties to funding WADA and domestic anti-doping tests. Although ratification of the Convention does not impose new costs upon the U.S. government, it would commit the United States to continuing such funding in the future. Some critics argue that the cost of anti-doping activities, especially legal costs of adjudicating doping cases, outweighs the benefits of deterring the use of performance-enhancing substances in sport. USADA's legal expenses, for example, represented more than 15% ($1.8 million) of its total expenses in 2006. Already in 2008, USADA has sought more than $1 million in external financial support from WADA for its legal case against U.S. cyclist Floyd Landis, who was stripped of the 2006 Tour de France title for a doping violation. | Summary: The International Convention Against Doping in Sport seeks to harmonize anti-doping commitments for non-professional sports at the international level. This Convention, adopted by the United Nations Educational, Scientific, and Cultural Organization (UNESCO) in 2005, entered into force on February 1, 2007. On July 21, 2008, the Senate approved the treaty for ratification (Treaty Doc 110-14), subject to an understanding, a declaration, and a condition. President George W. Bush signed the instrument of ratification for the treaty on August 4, 2008. Issues that may continue to arise as policymakers evaluate the treaty include its relationship to anti-doping regulations in professional sports and the legitimacy and effectiveness of current international anti-doping activities. U.S. ratification does not require changes to current federal laws. | 2,758 | 199 | gov_report | en |
Summarize: In the latest blunder for an Obama administration ambassadorial nominee, prolific Democratic campaign fundraiser Noah Mamet admitted Thursday that he has never been to Argentina – the nation where he wants to represent U.S. interests as America's top diplomat. The White House has tapped him to be the U.S. ambassador to the South American country. 'Mr. Mamet have you been to Argentina,' asked Florida Republican Sen. Marco Rubio during the Californian's confirmation hearing. SCROLL DOWN FOR VIDEO. Ambassador: Noah Mamet has never been to Argentina, but he's President Obama's pick to be the U.S.' next ambassador to the South American nation. 'I haven't had the opportunity yet to be there,' he admitted, ashen-faced. 'I've traveled pretty extensively around the world, but I haven't yet had the chance.' Mamet, like other ambassadorial nominees, raised copious amounts of campaign cash for President Obama – at least $500,000 during the 2012 campaign cycle, and another $500,000 or more in 2008 according to data from the Center for Responsive Politics. There's nothing new about fundraisers being rewarded with cushy posts overseas, but they usually have some genuine qualifications for directing U.S. foreign policy in the country where they end up. Mamet's embarrassing showing comes after a similar face-palm moment from George Tsunis in January who told Arizona Sen. John McCain that he, too, had never visited the country where he asked to be posted. 'Mr. Tsunis, have you been to Norway?' McCain asked. 'I have not,' Tsunis admitted. Norway: George Tsunis has never been to Norway, and doesn't know much about it - but he's our new Ambassador. The Long Island hotel magnate who has bundled and contributed at least $1.3 million to Obama and other Democrats, proceeded to deliver what one Scandinavian newspaper called a 'faltering, incoherent performance,' saying among other things that Norway has a 'president.' The nation is actually a constitutional monarchy, much like the United Kingdom, with both a King and a parliament. Tsunis also gaffed when he described the Progress Party, an anti-immigration political group with seven cabinet ministers among its members, as a part of 'fringe elements' that'spew their hatred.' 'Norway has been very quick to denounce them,' he said, before McCain corrected him and pointed out that they are an integral part of Norway's coalition government. Tsunis once contributed $50,000 to McCain's Senate campaign before defecting to the Democratic side and collecting more than $1 million to send Barack Obama to the White House. Hungary: Soap opera producer Colleen Bell doesn't know what the U.S. interests are in Hungary - yet, she's the new ambassador. He won Senate confirmation this week, as did Max Baucus, a former U.S. senator who is to be the U.S. ambassador to China. Faced with a tough question last week during his confirmation hearing about Chinese military provocation against Japan in the East China Sea, Baucus said plainly, 'I'm no expert on China.' The prize for ambassadorial cluelessness, though, may go to Colleen Bell, an Obama bundler who put $800,000 in play for Democrats. She's trading in her work as producer of the soap opera 'The Bold and the Beautiful' to become ambassador to Hungary. Asked by McCain to describe America's strategic interests in that nation, she launched into a rambling response worthy of a teen beauty pageant contestant. China: Newly crowned Ambassador to China Max Baucus freely admits that he's 'no real expert' on the Asian nation. 'Well, we have – our strategic interests, in terms of what are our key priorities in Hungary,' Bell began, 'I think our key priorities are to improve upon, as I mentioned, the security relationship and also the law enforcement and to promote business opportunities, increase trade... Our strategic interests are to work collaboratively as NATO allies.' 'To work to promote and protect the security, both – for both countries and for – and for the world,' she rambled on, 'to continue working together on the cause of human rights around the world, to build that side of our relationship while also maintaining and pursuing some difficult conversations that might be necessary in the coming years.' | Summary: Noah Mamet bundled at least $1 million in campaign donations to President Obama's two presidential election campaigns, and now he's in line to be the U.S. ambassador to Argentina. When Sen. Marco Rubio asked him during his confirmation hearing if he had ever visited the South American country, he admitted that he hadn't. Last month the Obama nominee for a similar post in Norway demonstrated a lack of knowledge about that nation's political structure and said he had never been there. Newly minted ambassador Max Baucus, now headed to Beijing, freely admitted in his own hearing that he's 'no real expert on China' The new ambassador to Hungary is a soap opera producer and prolific Democratic fundraiser who couldn't tell senators what America's strategic interests are in that country. | 1,011 | 177 | cnn_dailymail | en |
Summarize: FIELD OF THE INVENTION The present invention relates to an entertainment device and, more particularly, to a convertible, projected implement/target activity device, where the device includes a projectable implement and a target area and where, in one mode, the projected implement reaching the target area is thereafter contained by the entertainment device and, in a second mode, the projected implement reaching the target area is thereafter directed away from the entertainment device to encourage more active children to pursue and retrieve the projected implement. BACKGROUND Young children enjoy placing or throwing projectiles in defined areas such as holes, hoops or other types of open target areas. Children develop and become more mobile as they explore crawling, walking and other motor skills. At each stage of development, a child will be more agile and capable than in earlier stages of development. Parents want to encourage exploration at each developmental stage in order to assist in passage to the next developmental stage. To this end, reconfigurable entertainment devices offer parents an opportunity to encourage exploration at various developmental levels. Reconfigurable entertainment devices can provide skill level appropriate stimulation at one developmental stage and can then be reconfigured to provide appropriate stimulation at a more advanced skill level/developmental stage. In the present case, a reconfigurable childrens' projected implement/target activity device is disclosed. The device can be reconfigured into multiple configurations to stimulate children of different distinct skill and developmental levels. The device includes a graspable projectable implement, a target area and a projected implement movement controller. A child directs the implement through the target area after which the projected implement movement controller controls the movement of the implement. The projected implement can be a ball or any object that a child can grasp easily. The target area can be the open area of a ring, hoop, or other opening, through which the projected implement passes. The target area may be suspended above the projected implement movement controller. The projected implement movement controller may also function as a reversible base for the activity device. The projected implement movement controller of the present invention includes a first side and a second side. The first side of the projected implement movement controller has a concave shape and the second side has a convex shape. In a first configuration of the activity device of the present invention, the first side of the projected implement movement controller faces the target area so that a projected implement, passing through the target area, comes in contact with the projected implement movement controller. Because the first side of the projected implement movement controller is concave, when the projected implement passes through the target area, the projected implement is contained in the concave, bowl-shaped, side of the movement controller within proximity of the child. Alternatively, when the reversible projected implement movement controller is reconfigured to expose the movement controller's, second, convex side and the projected implement passes through the target area, the projected implement deflects off of the movement controller's dome-shaped, convex, surface and moves away from the activity device. The activity device according to the present invention therefore facilitates two modes of activity for children at different developmental levels. In the first mode where the concave, bowl-shaped, surface of the projected implement movement controller faces the target area, a younger, less mobile, child can place the implements through the target area and the movement controller will corral and contain the implements in close proximity to the child. This first mode also provides a convex surface pointing away from the target area and toward the supporting surface. In the first mode, the convex surface of the projected implement movement controller contacts the supporting surface to allow the activity device to rock back and forth as the child plays. In the second activity mode where the convex, dome-shaped surface of the projected implement controller faces the target area, the projected implements are deflected away from the activity device and must be retrieved as the child plays. This second activity mode therefore encourages children to be more active and further improves their motor skills and hand-eye coordination. The activity device of the present invention also provides sensory-stimulating rewards for a child successfully reaching the target area with a projected implement. An optical sensor may be utilized in the target area to sense the presence of the projected implement in the target area. Thus, the presence of the projected implement in the target area may trigger sensory-stimulating output from the activity device. The sensory-stimulating output may include lights, sound effects, speech, and/or music. Thus, a child that successfully reaches the target area with the projected implement is therefore rewarded with sensory-stimulating output to encourage continued play. Additionally, the activity device of the present invention could also incorporate a motion sensor to generate sensory-stimulating output at the slightest touch to further encourage continued play. SUMMARY Generally, the present invention device discloses a children's activity device comprising a projectable implement and a target area at which the implement is to be directed. The activity device includes a sensor that senses when the target area has been successfully reached by the projected implement and a sensory-stimulating output generating device that receives a signal from the sensor. When the sensory-stimulating output generating device receives the success signal from the sensor, it generates sensory-stimulating to encourage continued play. Specifically, the present invention discloses an activity device having a target area for receiving a plurality balls and an electronics unit including a sensor that detects the presence of a ball passing through the target area and a electronics controller that instructs the generation of sensory-stimulating output upon such detection. The present invention further contains a reconfigurable projected implement movement controller that directs and controls the movement of the projected implement after the target are has been successfully reached. The projected implement movement controller is reconfigurable in that one side of the projected implement movement controller is convex to direct a projected implement away from the activity device while the opposite side of the movement controller is concave to corral and contain the projected implement within the proximity of the activity device. The projected implement movement controller is connected to the target are such that, relative to the target area, the projected implement movement controller is reversible between the concave and convex sides. When the projected implement movement controller is oriented in the convex arrangement, balls passing through the target area, fall on the movement controller and are directed away from the activity device. Conversely, when the projected implement movement controller is reversed so that the concave side is directed upward, the balls passing through the target area are contained in the movement controller in close proximity to the activity device. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 illustrates a perspective view of a child playing with the activity device of the present invention, with the activity device shown in its containment mode. FIG. 2 illustrates an enlarged top perspective view of the concave side of the reversible projected implement movement controller/base of the activity device of FIG. 1, showing the base split into its two component parts. FIG. 3 illustrates an enlarged top perspective view of the convex side of the reversible projected implement movement controller/base of the activity device of FIG. 2. FIG. 4 illustrates an enlarged top perspective view of the concave side of the reversible projected implement movement controller/base of the activity device of FIG. 1 in its assembled form. FIG. 5 illustrates an enlarged top perspective view of the convex side of the reversible projected implement movement controller/base of the activity device of FIG. 1 in its assembled form. FIG. 6 illustrates an enlarged side perspective view of the reversible projected implement movement controller/base of the activity device of FIG. 1. FIG. 7 illustrates an enlarged side perspective view of the target area and target support arms of the activity device of FIG. 1. FIG. 8 illustrates an enlarged top perspective view of the target area and target support arms of the activity device of FIG. 1. FIG. 9 illustrates a close-up enlarged top perspective view of the target area of the activity device of FIG. 1. FIG. 10 illustrates an enlarged perspective view of the concave side of the reversible projected implement movement controller/base, the target area, and the target support arms of the activity device of FIG. 1. FIG. 11 illustrates manner of connection of the reversible projected implement movement controller/base and the target support arms of the activity device of FIG. 1 during assembly into the deflection mode. FIG. 12 illustrates an enlarged perspective view of the connection end of one of the target support arms of the activity device of FIG. 1. FIG. 13 illustrates an enlarged perspective view of one of the support arm reception slots of the reversible projected implement movement controller/base of the activity device of FIG. 1. FIG. 14 illustrates an enlarged perspective view showing the connection end of the support arm received in the guided reception slot of the activity device of FIG. 1. FIG. 15 illustrates an enlarged perspective view of the inner side of one of the support the activity device of FIG. 1. FIG. 16 illustrates an enlarged perspective view of the electrical contacts in another of the reception slots of the reversible projected implement movement controller/base of the activity device of FIG. 1. FIG. 17 illustrates an electronic schematic of the activity device of FIG. 1 in accordance with the present invention. FIG. 18 illustrates a perspective view of the activity device of the present invention showing the reversible projected implement movement controller/base holding two projectiles while configured in the containment mode. FIG. 19 illustrates an enlarged side perspective view of the activity device of FIG. 1 showing the reversible projected implement movement controller/base configured in the deflection mode. FIG. 20 illustrates an enlarged side perspective view of the activity device of FIG. 19 configured in the deflection mode and showing a projectile being deflected away from the activity device. Like reference numerals have been used to identify like elements throughout this disclosure. DETAILED DESCRIPTION In accordance with the present invention, an activity entertainment device 100 is disclosed. The activity device 100 is a reconfigurable to allow for two different modes of activity. In a containment mode, the activity device 100 contains or corrals the projected implements that have passed through the target area to accommodate less mobile/younger children. Alternatively, in a second, deflection mode, projected implements that pass through the target area are deflected away from the activity device 100, requiring the child to retrieve the projected implements and thereby encouraging retrieval activity. In addition, in the containment mode, the portion of the base of the activity device 100 that is in contact with the supporting surface is convex to allow for the rocking of the activity device 100. In the deflection mode, the portion of the base of the activity device 100 that is in contact with the supporting surface is concave and thus, a stable, non-rocking, characteristic is achieved. FIG. 1 illustrates a perspective view of a child playing with the activity device 100 of the present invention, with the activity device 100 shown in its containment mode. As shown, the activity device 100 is a drop/toss toy with a hoop-type target portion 110 that senses a projectile 130 passing through the target portion 110 and generates music to reward the child when a projectile 130 such as a ball is tossed through the target portion 110. The activity device 100 generally comprises a target portion 110 formed as a hoop or ring, a bowl shaped reversible base 150 for directing the projectile after passing through the target portion 110, support arms 120 and 140 for supporting the target portion 110 above the reversible base 150 and projectiles 130. In the containment mode, the reversible base 150 corrals the projectiles 130 that have passed through the target portion 110. As illustrated, in the containment mode, the convex portion of the reversible base 150 is in contact with the supporting surface 160 to provide a rocking motion for the activity device 100. FIG. 2 illustrates an enlarged top perspective view of the concave side of the reversible projected implement movement controller/base of the activity device of FIG. 1, showing the base split into its two component parts. In order to reduce the size of the retail packaging (not shown) for the activity device 100 of the present invention, the reversible base 150 is constructed from two separate interlocking portions ( 210 and 240 ). Portion 210 includes of a female receptacle 212. Female receptacle 212 is designed to receive key 246 on portion 240. Portion 210 also includes a plurality of fastener tabs 214, 215, 216 with apertures therein. The fastener tabs 214, 215, 216 extend from the side of portion 210. Portion 240 contains a series of fastener-receiving recesses 341, 342, 343 (best seen in FIG. 3 ). Each fastener-receiving recess 341, 342, 343 is adapted to mate with a corresponding fastener tab 214, 215, 216 on portion 210 and receive a fastener. The female receptacle 212, key 246, fastener tabs 214, 215, 216, and fastener-receiving recesses 341, 342, 343 provide a simple, stable way to secure the portions 210 and 240 of the the reversible base 150 together after removal from the retail packaging (not shown). To secure the portions 210 and 240 together, portion 240 is held above the portion 210 so that fastener tab 214 is aligned with fastener-receiving recess 341, fastener tab 215 is aligned with fastener-receiving recess 342, and fastener tab 216 is aligned with fastener-receiving recess 343. Portion 240 is then lowered so that the corresponding fastener tabs fit snuggly within the corresponding fastener-receiving recesses. The female receptacle 212 and the key 246 will obviously also align and fit snuggly together. Portion 210 can then be secured to the portion 240 by directing fasteners through a apertures in the fastener tabs 214, 215, 216, into the corresponding fastener-receiving recesses 341, 342, 343. The heads of the fasteners may be countersunk into the fastener tabs 214, 215, 216 so that they do not protrude above the surface on the convex side 330 of the reversible base 150. As shown in FIGS. 2-5, the reversible base 150 includes looped members 221 and 222 that form support arm reception slots 231 and 232 for receiving portions of support arms 120 and 140. As shown in FIG. 2, the reversible base 150 has a swirl pattern 228 molded into the concave surface of the containment side 220 of the reversible base 150. Additionally, portion 240 of the reversible base 150 includes an arcuate opening 245 for easy removal of the projectiles 130 from the reversible base 150 during play. FIG. 3 shows a battery compartment door 333 on the convex side 330 of the reversible base 150. The battery compartment door 333 covers a compartment area where the batteries that power the activity device 100 located. A countersunk fastener secures the door 333 in a closed position so that neither the door 333 or the fastener protrude above the convex side 330 of the reversible base 150. FIGS. 4-6 show the reversible base 150 in its assembled form. FIG. 4 illustrates an enlarged top perspective view of the concave side 220 of the reversible projected implement movement controller/base 150 of the activity device 100 of FIG. 1 in its assembled form. FIG. 5 illustrates an enlarged top perspective view of the convex side 330 of the reversible projected implement movement controller/base 150 of the activity device 100 of FIG. 1 in its assembled form. FIG. 5 also shows a plurality of fastener apertures and fasteners therein to secure the upper and lower portions of the reversible projected implement movement controller/base 150 together. FIG. 6 illustrates an enlarged side perspective view of the reversible projected implement movement controller/base 150 of the activity device 100 of FIG. 1 FIG. 7 illustrates an enlarged side perspective view of the target portion 110 and target support arms 120, 140 of the activity device 100 of FIG. 1. As discussed briefly above, the activity device 100 of the present invention has a hooped or ringed target portion 110 that is supported above the reversible base 150 by support arms 120 and 140. The upper portion of the hoop is composed of two opaque portions 753, 754 and two translucent portions 752, 756. The target portion 110 houses electronic components that produce light which shines from the translucent upper portions 752, 756 of the target portion 110. A fabric net 758 is suspended from the inside of the target portion 110 to create a basketball style activity. Support arms 120 and 140 extend from a lower portion 719 of the target portion 110 and extend downward. Support arm 140 includes electronic components (e.g., wiring) associated with power, sound and light. Support arm 140 also houses the power/volume switch 715 on the outside surface of the arm 140 and contains apertures (best seen in FIG. 15 ) through which sound, generated by a speaker passes. The electronic features of the activity device 100 of the present invention will be explained in more detail below. Support arm 140 also supports two mechanical activity rollers 711 and 712. The rollers provide additional entertainment value and are also intended to improve a child's manual dexterity. Both support arms 120 and 140 may include an external raised design that is molded into the arm. In the illustrated embodiment, the raised design is stylized as a serpentine vine with leaves. The lower end of support arms 120 and 140 may be mechanically and electronically connected to the reversible base 150. Details of the connection of the support arms 120 and 140 to the reversible base 150 will be discussed in more detail below. Support arm 120 extends from an upper end that is attached to the lower portion 719 of the target portion 110 down to a lower end that also is connectable to the reversible base 150. The support arm 120 does not contain any electronic elements and is generally hollow. Stiffening ribs 725 extend along the length and width of the arms 120 and 140 to minimize the amount of material necessary while maintaining the structural rigidity of the arms 120 and 140. An animal-styled mechanical spinner 721 is supported on the outer side of support arm 120 to perform cartwheels when batted by a child. The spinner 721 is connected to and supported on a projection 727 that is rotatably secured in the support arm 120. Like support arm 140, the lower portion of support arm 120 is connectable to the reversible base 150, which connection will be described below in more detail. FIGS. 8 and 9 also illustrate enlarged images of the support arms 120 and 140 as well as the target portion 110. FIGS. 8 and 9 also show the sensor transmitter 860. The sensor receiver 862 is located on the opposite side of the target portion 110 from the sensor transmitter 360. In the illustrated embodiment, the sensor transmitter/receiver 860, 862 is an optical sensor. A beam of light is directed from the transmitter 860 across the opening 880 in the target portion 110 to the receiver 862. Obviously, the positions of the sensor's transmitter 860 and receiver 862 can be reversed. When a projectile/implement 130 (see FIG. 1 ) passes through the opening 880 in the target portion 110, it interrupts the beam of light passing from the transmitter 860 across the opening 880 in the target portion 110 to the receiver 862 which sends a signal to a sensory-output generating device. The sensory-output generating device then generates sensory output to reward the child for placing or tossing the projectile/implement 130 into the opening 880 in the target portion 110. The operation of the electronic components of the activity device 100 of the present invention will be discussed in more detail below. FIG. 10 illustrates an enlarged perspective view of the concave side 220 of the reversible projected implement movement controller/base 150, the target portion 110, and the target support arms 120, 140 of the activity device 100 of FIG. 1. After assembly of the two portions 210 and 240 of the reversible projected implement movement controller/base 150, the basic assembly of the activity device 100 is complete. Disassembling the activity device 100 and reassembling the activity device 100 between the containment mode and the deflection mode requires only reversing the base 150 which does not require the use of any fasteners or tools. Thus, reconfiguration between the containment mode and the deflection mode amounts to not much more than a plug-in/plug-out type of exercise. FIG. 10 shows the assembled base 150 of the activity device 100 device ready to be assembled into either the containment mode or the deflection mode. Specifically, when the base 150 of the activity device 100 is assembled in the orientation shown in FIG. 10, the result is a completely assembled activity device 100 in the containment mode in which projectiles/implements 130 are collected in the concave side 220 of the base 150 after passing through the target portion 110. FIG. 11 illustrates manner of connection of the reversible projected implement movement controller/base 150 and the target support arms 120, 140 of the activity device 100 of FIG. 1 during assembly into the deflection mode. Specifically, when the activity device 100 is assembled in the orientation shown in FIG. 11, the result is a fully assembled activity device 100 in the deflection mode. To assemble the activity device 100 in the deflection mode, the lower connection ends 1114, 1124 of the support arms 140, 120 are vertically aligned with their corresponding support arm reception slots 231 and 232 in the base 150. The connection ends 1114, 1124 are lowered and slid into and received by the support arm reception slots 231, 232. The connection ends 1114, 1124 slide into the support arm reception slots 231, 232 until they reach end stops 1113 and 1123. The connection between the support arms 120, 140 and the reversible base 150 will now be described in detail along with FIGS. 12-14. Because support arm 140 contains electronic components and support arm 120 does not, the support arms are not interchangeable within the support arm reception slots 231 and 232 in the base 150. In other words, connection ends 1114 must be received into reception slot 231 and connection end 1124 must be received into reception slot 232. To ensure that connection ends 1114 and 1124 are received only in the correct reception slots and to insure reception into the reception slots 231 and 232 with precise alignment, the connection end 1124 of the support arm 120 has guide members 1224. Guide member 124 (shown in FIG. 12 ) is a groove in the outwardly facing surface of the connection end 1124 of support arm 120. As shown in FIG. 13, complementarily guide member 1326 is a longitudinal projection on the inside of looped member 222 projecting into reception slot 232 towards the center of the activity device 100. FIG. 14 shows connection end 1124 of support arm 120 partially inserted into reception slot 232 of the looped member 222. During insertion, guide member 1326 of the looped member 222 slides within grooved member 1224 of the connection end 1124 of support arm 120 to ensure proper alignment between the connection end 1124 and the reception slot 232. The connection end 1124 slides easily into the reception slot 232 until end stop 1123 prevents further insertion. FIGS. 15 and 16 illustrate how electrical contact is maintained between the portion of the electronic system within the reversible base 150 and the remainder of the electrical system within the support arm 140 and the target portion 110. FIG. 15 also illustrates a hole pattern 1510 on the inner surface of support arm 140. Hole pattern 1510 covers a sound producing speaker. The connection end 1114 of support arm 140 contains an inside electrical contact 1516 and an electrical projection contact 1517 both on the inside surface of the connection end 1114 of support arm 140. Contacts 1516 and 1517 conduct electrical current between the reversible base 150 and the target portion 110. Correspondingly and as illustrated in FIG. 16, the inside surface of the reception slot 231 has three reception electrical contacts 1610, 1611, and 1612 for receiving the inside electrical contact 1516 and the outside electrical contact 1517. The inside 1516 and outside 1617 electrical contacts are spring loaded so that they retract into the inner surface of the connector end 1114 when the connector end 1114 is being inserted into the reception slot 231. This retraction prevents the electrical contacts 1516, 1517 from becoming an obstacle to insertion of the connection end 1114 into 231. The electronics assembly of the activity device 100 of the present invention can also identify the orientation of the reversible base 150 and thus the mode (containment or deflection) in which the activity device 100 is operating. Appropriate sensory-stimulating output can then be generated depending on the mode in which the activity device 100 is operating. Specifically, the activity device 100 can determine the mode because the inner electrical contact 1516 is always aligned with the central reception electrical contact 1611. However, the outside electrical contact 1517 is aligned with one of the outer reception electrical contacts 1610 in the containment mode and the other of the outside outer reception electrical contacts 1612 in the deflection mode when the base 150 is reversed. The orientation of the reversible base 150 may therefore be determined by detecting which of the outer reception electrical contacts 1610 or 1612 receives the outer electrical contact 1517. Again, these electrical contacts 1516, 1517, and 1610 - 1612 allow power and electrical signals to be passed between the power source and the electronics controller (housed in the base 150 ) to the speaker, lights, and receiver/transmitter (all of which are located in the support arms 120, 140 and the target portion 110 ) without the use of wires extending out of the base 150. As discussed above, the activity device 100 of the present invention may include one or more electronic components. FIG. 17 illustrates an electronic schematic of the activity device 100 of FIG. 1 in accordance with the present invention. In the illustrated embodiment, the electronics assembly 1700 includes an optical sensor 1710. Specifically, the sensor of the electronics assembly 1700 includes an LED emitter (light emitting portion/transmitter) 860 and a corresponding photoconductive receiver (light receiving portion) 862 (e.g., where the light emitting portion and the light receiving portion makes up a “sensor pair”). The electronics assembly 1700 also includes two lights generators 1750, 1752. The light generators 1750, 1752 generally flash to the beat of the music. Light generators 1750, 1752 are housed beneath the two translucent portions 752, 756 of the target portion 110. The flashing lights 1750, 1752 act as a reward for various encouraged behavior. As discussed below, a number of events trigger a light display response in a number of different modes. The electronics assembly 1700 may further include a speaker 1760 coupled to both a microprocessor/electronics controller 1780 and the power source 1770. The electronics assembly 1700 further includes three switches, each switch being associated with a particular feature of the activity device 100. Switch 1720 A, 1720 B is responsible for controlling power and volume options (switch 1720 A and 1720 B are simply illustrated as two poles of a single switch). Switch 1720 A, 1720 B may be used to control the connection of a power source 1770 to the electronics assembly 1700 (turning it on and off). The power source 1770 may include, for example, three “AAA” batteries. The schematic of FIG. 17 shows electrical contacts 1, 2 and 4 separate from contacts 8, 7 and 5, however, these contacts all belong to the same switch and are all controlled by power/volume (illustrated as switch 715 in FIG. 7 ). Therefore, when switch 1720 A, 1720 B is in position ( 1, 8 ), no battery power is available to the controller 1780. In position ( 2, 7 ), the battery power is available to the controller 1780 and a low sound is generated by the speaker 1760. Finally, in position ( 4, 5 ), power is available to the circuit and full sound is generated by the speaker 1760. When engaged in either of the second or third positions (“low”, “high”), the switch 1720 A, 1720 B communicates with the microprocessor 1780, and switch-specific sensory output (sounds and/or lights) is generated. A second internal switch 1730 may be included for additional functionality (such as a motion sensor housed within base 150 ). After the first switch 1720 A, 1720 B is activated, and power is available to the circuit, the controller unit 1780 illuminates lights 1750 and sounds before transferring to a sleep mode. The controller unit 1780 enters a sleep mode in which any further movement triggers lights and sounds. A third switch 1740 may be used to activate a “Try-Me” mode. The microprocessor controller unit 1780 has the “Try-Me” mode that can be activated when the product is still in the package on the retailer's shelf. In other words the shopper can activate the microprocessor unit 1780 to initiate a limited sample of the sounds and lights that would be generated in normal modes. When the packaging is removed the “Try-Me” mode may be disabled. As noted above, each of the speaker 1760, the power source 1770, the light emitter 860 the light receiver 862, the switches 1720 A-B, 1730, 1740, and the lights 750 are operatively coupled (connected) to the microprocessor unit 1780. The type of microprocessor is not limited, and includes microcontrollers, microprocessors, and other integrated circuits. Microprocessor unit 1780 recognizes and controls signals generated by and to the light emitter 860, the light receiver 862, the various switches 1720 A-B, 1730, 1740, and the lights 750. In addition, microprocessor unit 1780 generates and controls operational output. The microprocessor unit 1780 continually monitors the electronic status of the light emitter 860, the light receiver 862 and the switches 1720 A-B, 1730, and 1740, generating and altering the sensory output (e.g., sounds and/or lights) accordingly. The operation of the activity device 100 will now be described. In operation, when the first switch 715 (internally, switch 715 is schematically illustrated as switch 1720 A-B) is engaged, power is sent from the power source 1770 to the microprocessor unit 1780. Once powered and active, the microprocessor unit 1780 of the activity device 100 is in the start-up mode. In the start up mode, the microprocessor unit 1780 activates lights from the light sources 1750 and sounds from the speaker 1760 for a predetermined period of time. The microprocessor unit 1780 then changes to beam break mode. In beam break mode, the emitter 860 and the receiver 862 of the sensor 1710 in the target portion 110 is activated. If a ball/implement 130 passing through the target portion 110 breaks the beam, the microprocessor unit 1780 activates sounds through speaker 1760 and lights 1750 blink to the music. If the beam is not broken for a predetermined period of time (e.g., one minute), the microprocessor unit 1780 goes into “sleep” mode. In sleep mode, the beam break feature is turned off and the internal motion sensor 1730 feature (if present) may be activated. Whenever the activity device 100 is disturbed to activate motion sensor 1730, the microprocessor unit 1780 goes back to the start-up mode, generates sounds and flashing coordinated lights for a period of time, turns the beam break feature on and waits for the beam sensor 1710 to be broken by a ball/implement 130. FIGS. 18-21 show the fully assembled activity device 100 in its various modes. FIG. 18 shows the activity device 100 in its containment mode with two balls/implements 130 that have passed through the target portion 110 and been contained in the concave surface 220 reversible base 150. As a child puts the balls/implements 130 through the target portion 110, the sensor beam is broken to activate sounds and lights before the balls/implements 130 are contained in the concave surface 220 reversible base 150. In this mode, the activity device 100 also rocks back and forth on the convex outer surface 330 of the reversible base 150. FIGS. 19-20 show the activity device 100 in the fully assembled deflection mode. In this deflection mode the convex surface 330 of the reversible base 150 faces the target portion 110. The portion of the reversible base 150 contacting the supporting surface 160 is stable and thus, the activity device does not rock in the deflection mode. When balls/implements 130 pass through the target portion 110 and break the sensor beam, the microprocessor unit 1780 generates lights and sounds. The balls/implements 130 then drop onto the convex surface 330 of the reversible base 150 and are deflected away from the activity device 100. The child can then chase and retrieve the balls/implements 130 before placing them in the target portion 110 again. FIG. 20 shows a ball/implement 130 in multiple positions as the ball contacts the convex surface 330 of the reversible base 150 and is directed away from the activity device 100. The electronics assembly 1700 in accordance with the present invention may include any combination of sensors, switches, lights, speakers, animated members, motors, and sensory output generating devices. The microprocessor unit 1780 may produce any combination of audio and visual effects including, but not limited to, animation, lights, and sound (music, speech, and sound effects). The output pattern is not limited to that which is discussed herein and includes any pattern of music, lights, and/or sound effects. The electronics assembly 1700 may also include additional switches or sensors to provide additional sensory output activation without departing from the scope of the present invention. Thus, it is intended that the present invention cover the modifications and variations of this invention that come within the scope of the appended claims and their equivalents. For example, it is to be understood that terms such as “left”, “right” “top”, “bottom”, “front”, “rear”, “side”, “height”, “length”, “width”, “upper”, “lower”, “interior”, “exterior”, “inner”, “outer” and the like as may be used herein, merely describe points of reference and do not limit the present invention to any particular orientation or configuration. | Summary: A reconfigurable target/projectile activity entertainment device is disclosed, wherein the device includes a projectile, a target having a target area and a reversible base connectable to the target. The target is in the form of a hoop or ring and is disposed above the reversible base. The hoop has an opening therein that forms the target area. The hoop contains a sensor that detects a projectile passing through the target area and communicates with a sensory generator to generate sensory-stimulating output (i.e., lights and sounds). Projectiles directed through the target area drop to the reversible base below the target area. The reversible base has a first side and a second side. The first side is concave for collecting a projectile that drops thereon. The second side is opposite the first side and has a convex side that deflects projectiles dropping thereon to deflect the projectile away from the device. The reversible base can be reconfigured between a first mode wherein the concave side faces the target area and a second mode that wherein the convex side faces the target. | 9,319 | 239 | big_patent | en |
Summarize: A flagship scheme launched by David Cameron to get three million people texting or emailing their blood pressure and other vital signs to their GP surgery has been quietly dropped. When the Prime Minister announced the initiative in 2011, he said ‘telehealth’ had been found to be a ‘huge success’ – and promised three million people would use it within five years. He claimed it would save the NHS £1.2billion a year by keeping patients with long-term conditions like heart disease and diabetes out of hospital. Ill health: Many older people were put off by David Cameron's modern 'telehealth' gizmos. Getting patients to monitor themselves – for example, taking blood pressure or blood sugar readings – and send the information remotely would make them take better care of their own health, Ministers believed. And the NHS would save more money by not having to provide so many routine appointments. ‘This is going to make an extraordinary difference to people,’ Mr Cameron predicted. But now the ‘3millionlives’ project has been shelved, after subsequent research found NHS telehealth programmes to be expensive and largely ineffective. Many older people were put off by having to use modern gizmos to send in their details, researchers found. Nurses were enthusiastic, as the scheme cut their workload and ‘empowered’ patients to care for themselves. But doctors complained they were inundated by a ‘tsunami’ of patient data. And last month, academics found the extra £1,014 a year it cost to provide each patient with the monitoring service and equipment did not cut deaths or rates of admission to care homes. When the Prime Minister announced the initiative, he said ‘telehealth’ had been found to be a ‘huge success’ Last night, GPs said politicians should concentrate on getting the basics right, rather than obsessing about ‘gadgets’. Dr Margaret McCartney, who presents the BBC Radio 4 programme Inside Health, said: ‘This expensive black box kit isn’t good value for money – and there is no gadget in the world that can help a sick person to eat, wash and dress. ‘NHS England needs to invest in what works – especially hands-on human care supplied by carers who currently often don’t have enough time to supply people’s basic needs.’ But Professor James Barlow from Imperial College, London, who has helped evaluate the programmes, believes telehealth still has a future. He said: ‘The evidence is that telehealth does lead to lower rates of admission and speedier discharge.’ The 3millionlives initiative has now been rebranded, with NHS England officials referring to it as TECS (Technology Enabled Care Services), but with no ambitious targets attached. Professor Sir Bruce Keogh, medical director of NHS England, has written to health bosses asking them to support the revamped scheme. Advocates of telehealth argue the popularity of smartphone health apps – which measure everything from sleep to fitness levels – make it more likely ‘remote care’ will become increasingly accepted. A spokesman for NHS England claimed their ambition was still to help three million people with long-term conditions using remote care devices. He added: ‘Twenty years from now, we will use technology to access our health services as a matter of course.’ | Summary: David Cameron announced flagship 'telehealth' initiative back in 2011. Said it would save NHS £1.2billion a year and keep patients out of hospital. Got patients to monitor themselves and email results to their doctor. But the programmes have been found to be expensive and ineffective. Older people were put off by having to use modern gizmos to send in details. GPs say politicians should concentrate on getting the basics right. | 707 | 98 | cnn_dailymail | en |
Write a title and summarize: SECTION 1. SHORT TITLE. This Act may be cited as the ``Medicare Enhancements for Needed Drugs Act of 2004''. SEC. 2. GAO STUDIES AND REPORTS ON PRICES OF PRESCRIPTION DRUGS. (a) Review and Reports on Retail Prices of Prescription Drugs.-- (1) Initial review.--The Comptroller General of the United States shall conduct a review of the retail cost of prescription drugs in the United States during 2000 through 2003, with an emphasis on the prescription drugs most utilized for individuals age 65 or older. (2) Subsequent review.--After conducting the review under paragraph (1), the Comptroller General shall continuously review the retail cost of such drugs through April 1, 2006, to determine the changes in such costs. (3) Reports.-- (A) Initial review.--Not later than September 1, 2004, the Comptroller General shall submit to Congress a report on the initial review conducted under paragraph (1). (B) Subsequent review.--Not later than July 1, 2005, January 1, 2006, and July 1, 2006, the Comptroller General shall submit to Congress a report on the subsequent review conducted under paragraph (2). (b) Annual GAO Study and Report on Retail and Acquisition Prices of Certain Prescription Drugs.-- (1) Ongoing study.--The Comptroller General of the United States shall conduct an ongoing study that compares the average retail cost in the United States for each of the 20 most utilized prescription drugs for individuals age 65 or older with-- (A) the average price at which private health plans acquire each such drug; (B) the average price at which the Department of Defense under the Defense Health Program acquires each such drug; (C) the average price at which the Department of Veterans Affairs under the laws administered by the Secretary of Veterans Affairs acquires each such drug; and (D) the average negotiated price for each such drug that eligible beneficiaries enrolled in a prescription drug plan under part D of title XVIII of the Social Security Act, as added by section 101 of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173), that provides only basic prescription drug coverage have access to under such plans. (2) Annual report.--Not later than December 1, 2007, and annually thereafter, the Comptroller General shall submit to Congress a report on the study conducted under paragraph (1), together with such recommendations as the Comptroller General determines appropriate. SEC. 3. INCLUSION OF AVERAGE AGGREGATE BENEFICIARY COSTS AND SAVINGS IN COMPARATIVE INFORMATION FOR BASIC MEDICARE PRESCRIPTION DRUG PLANS. Section 1860D-1(c)(3) of the Social Security Act, as added by section 101 of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173), is amended-- (1) in subparagraph (A)-- (A) in the matter preceding clause (i), by striking ``subparagraph (B)'' and inserting ``subparagraphs (B) and (C)''; and (B) by adding at the end the following new clause: ``(vi) Average aggregate beneficiary costs and savings.--With respect to plan years beginning on or after January 1, 2007, the average aggregate costs, including deductibles and other cost-sharing, that a beneficiary will incur for covered part D drugs in the year under the plan compared to the average aggregate costs that an eligible beneficiary with no prescription drug coverage will incur for covered part D drugs in the year.''; and (2) by adding at the end the following new subparagraph: ``(C) Average aggregate beneficiary costs and savings information only for basic prescription drug plans.--The Secretary shall not provide comparative information under subparagraph (A)(vi) with respect to-- ``(i) a prescription drug plan that provides supplemental prescription drug coverage; or ``(ii) a Medicare Advantage plan.''. SEC. 4. NEGOTIATING FAIR PRICES FOR MEDICARE PRESCRIPTION DRUGS. (a) In General.--Section 1860D-11 of the Social Security Act, as added by section 101 of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173), is amended by striking subsection (i) (relating to noninterference) and by inserting the following: ``(i) Authority To Negotiate Prices With Manufacturers.--In order to ensure that beneficiaries enrolled under prescription drug plans and MA-PD plans pay the lowest possible price, the Secretary shall have authority similar to that of other Federal entities that purchase prescription drugs in bulk to negotiate contracts with manufacturers of covered part D drugs, consistent with the requirements and in furtherance of the goals of providing quality care and containing costs under this part.''. (b) Effective Date.--The amendment made by this section shall take effect as if included in the enactment of section 101 of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173). SEC. 5. DISALLOWANCE OF DEDUCTION FOR ADVERTISING EXPENDITURES OF TAXPAYERS WHO DISCRIMINATE AGAINST FOREIGN SELLERS OF PRESCRIPTION DRUGS TO DOMESTIC CONSUMERS. (a) General Rule.--Part IX of subchapter B of chapter 1 of the Internal Revenue Code of 1986 (relating to items not deductible) is amended by adding at the end the following new section: ``SEC. 280I. ADVERTISING EXPENDITURES OF TAXPAYERS WHO DISCRIMINATE AGAINST FOREIGN SELLERS OF PRESCRIPTION DRUGS TO DOMESTIC CONSUMERS. ``(a) In General.--No deduction otherwise allowable under this chapter shall be allowed for any amount paid or incurred for advertising for the taxable year by any taxpayer who at any time during such taxable year discriminates against a qualified pharmacy or qualified wholesaler in the sale of prescription drugs. ``(b) Advertising.--For purposes of this section, the term `advertising' includes direct to consumer advertising and any activity designed to promote the use of a prescription drug directed to providers or others who may make decisions about the use of prescription drugs (other than the provision of free samples). ``(c) Qualified Pharmacy; Qualified Wholesaler.--For purposes of this section-- ``(1) Qualified pharmacy.--The term `qualified pharmacy' means any pharmacy located outside the United States which sells prescription drugs to consumers living in the United States. ``(2) Qualified wholesaler.--The term `qualified wholesaler' means any wholesaler located outside the United States which sells prescription drugs to entities selling prescription drugs to consumers living in the United States. ``(d) Discrimination.--For purposes of subsection (a), a taxpayer shall be treated as discriminating against a qualified pharmacy or qualified wholesaler in the sale of prescription drugs if such taxpayer publicly, privately or otherwise refuses to do business with a person or entity on the basis that the person or entity will pass along discounts offered to the person or entity to consumers living in the United States.''. (b) Clerical Amendment.--The table of sections for part IX of subchapter B of chapter 1 of such Code is amended by adding at the end thereof the following new item: ``Sec. 280I. Advertising expenditures of taxpayers who discriminate against foreign sellers of prescription drugs to domestic consumers.''. (c) Effective Date.--The amendments made by this section shall apply to taxable years beginning after the date of the enactment of this Act. SEC. 6. COST CONTAINMENT INCENTIVES. (a) In General.--Section 1860D-42 of the Social Security Act, as added by section 101 of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173), is amended by adding at the end the following new subsection: ``(c) Incentives to PDP Sponsors To Negotiate Lower Prices.-- ``(1) Authority.--The Secretary is authorized to provide incentive payments to PDP sponsors offering prescription drug plans that provide enrollees with access to negotiated prices used for payment of covered part D drugs under the plans that on average are not more than 10 percent greater than the lesser of-- ``(A) the average price at which the Department of Defense under the Defense Health Program acquires such drugs; or ``(B) the average price at which the Department of Veterans Affairs under the laws administered by the Secretary of Veterans Affairs acquires such drugs. ``(2) Information from va and dod.--Upon request of the Secretary of Health and Human Services, the Secretary of Defense and the Secretary of Veterans Affairs shall make available to the Secretary of Health and Human Services such information regarding acquisition prices of prescription drugs as the Secretary of Health and Human Services determines is necessary to conduct the incentive payment program under this subsection. ``(3) Application.--No incentive payments may be made under this subsection except pursuant to an application that is submitted and approved in a time, manner, and form specified by the Secretary. ``(4) Funding.--There shall be available to the Secretary from the MA Regional Plan Stabilization Fund under section 1858(e) during the period beginning on January 1, 2007, and ending on December 31, 2013, a total of $500,000,000 for making incentive payments under this subsection. ``(5) Annual reports.--For each year in which an incentive payment is awarded under this subsection, the Secretary shall submit a report to Congress containing a description of the operation of the incentive payment program.''. (b) Stabilization Fund Amendments.--Section 1858(e)(1) of the Social Security Act, as added by section 221(c) of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173), is amended-- (1) in the matter preceding subparagraph (A), by striking ``2'' and inserting ``3''; and (2) by adding at the end the following new subparagraph: ``(C) PDP incentive payments.--To provide incentive payments to PDP sponsors pursuant to section 1860D- 42(c).''. (c) Effective Date.--The amendments made by this section shall take effect as if included in the enactment of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173). SEC. 7. NAIC REVIEW AND REPORT ON CHANGES IN MEDIGAP POLICIES THAT PROVIDE COVERAGE OF PRESCRIPTION DRUGS CONTAINED IN THE MEDICARE PRESCRIPTION DRUG, IMPROVEMENT, AND MODERNIZATION ACT OF 2003. (a) In General.--The Secretary shall request the National Association of Insurance Commissioners to conduct a review of the changes to the rules relating to medicare supplemental policies that provide prescription drug coverage contained in subsection (v) of section 1882 of the Social Security Act (42 U.S.C. 1395ss), as added by section 104(a) of the Medicare Prescription Drug, Improvement, and Modernization Act of 2003 (Public Law 108-173). (b) Impact on Medicare Beneficiaries.--The review conducted pursuant to subsection (a) should focus on the impact the changes described in such subsec | Title: A bill to reduce the costs of prescription drugs for medicare beneficiaries, and for other purposes Summary: Medicare Enhancements for Needed Drugs Act of 2004 - Directs the Comptroller General to review and report to Congress on the retail cost of prescription drugs in the United States during 2000 and 2003 with an emphasis on the prescription drugs most utilized for individuals age 65 or older. Requires the Comptroller General, after conducting such review, to review continuously the retail cost of such drugs through April 1, 2006, to determine the changes in such costs. Requires the Comptroller General to conduct an ongoing study, for annual reports to Congress, that compares the average retail cost in the United States for each of the 20 most utilized prescription drugs for individuals age 65 or older with: (1) the average price at which private health plans acquire each such drug; (2) the average price at which the Department of Defense under the Defense Health Program acquires each such drug; (3) the average price at which the Department of Veterans Affairs under the laws administered by the Secretary of Veterans Affairs acquires each such drug; and (4) the average negotiated price for each such drug that eligible beneficiaries have access to under a Medicare prescription drug plan that provides only basic prescription drug coverage. Amends title XVIII (Medicare) of the Social Security Act (SSA) to include in the comparative plan information for beneficiaries under new Medicare part D (Voluntary Prescription Drug Benefit Program) a comparison of average aggregate prescription drug plan beneficiary costs and savings with respect to covered part D drugs with such costs for the same drugs for a beneficiary with no prescription drug plan. Repeals the prohibition against interference by the Secretary with the negotiations between drug manufacturers and pharmacies and prescription drug plan sponsors and the requirement of a particular formulary to institute a price structure for the reimbursement of Medicare part D covered drugs. Authorizes the Secretary instead, like other Federal entities that purchase prescription drugs in bulk, to negotiate contracts with manufacturers of covered part D drugs. Amends the Internal Revenue Code to disallow a tax deduction for advertising expenditures of taxpayers who discriminate against foreign sellers of prescription drugs to domestic consumers. Amends SSA title XVIII to authorize the Secretary to provide incentive payments out of the Medicare Advantage Regional Plan Stabilization Fund to sponsors offering prescription drug plans that provide enrollees with access to negotiated prices for payment of covered Medicare part D drugs. Requires such prices to be on average not more than ten percent greater than the lesser of: (1) the average price at which the Department of Defense under the Defense Health Program acquires such drugs; or (2) the average price at which the Department of Veterans Affairs acquires such drugs. Requires the Secretary to request the National Association of Insurance Commissioners to review and report to Congress on the changes to the rules relating to Medicare supplemental policies that provide prescription drug coverage under new Medicare part D. | 2,764 | 636 | billsum | en |
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